Alleviation of the allergenic potential of airborne and contact allergens by thioredoxin

ABSTRACT

Thioredoxin, a small dithiol protein, is a specific reductant for allergenic proteins and particularly allergenic proteins present in pollen and animal and plant sources. All targeted proteins contain disulfide (S—S) bonds that are reduced to the sulfhydryl (SH) level by thioredoxin. The proteins are allergenically active and less digestible in the oxidized (S—S) state. When reduced (SH state), they lose their allergenicity and/or become more digestible. Thioredoxin achieved this reduction when activated (reduced) either by NADPH via NADP-thioredoxin reductase (physiological conditions) or by lipoic acid chemical reductant. Skin tests carried out with sensitized dogs showed that treatment of the pollens with reduced thioredoxin prior to injection eliminated or decreased the allergenicity of the pollen. Studies showed increased digestion of the pollen proteins by pepsin following reduction by thioredoxin. Pollen proteins that have been reduced by thioredoxin are effective and safe immunotherapeutic agents for decreasing or eliminating an animal&#39;s allergic reaction that would otherwise occur upon exposure to the non-reduced pollen protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a CONTINUATION application of U.S. patentapplication Ser. No. 09/238,379 filed Jan. 27, 1999 now U.S. Pat. No.6,555,116; which is a Continuation-in-Part of U.S. application Ser. No.08/953,703 filed Oct. 17, 1997 now U.S Pat. No. 5,952,034, which arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the use of thiol redox proteins toreduce seed proteins such as cereal proteins, and to reduce enzymeinhibitor proteins, venom toxin proteins, pollen proteins and theintramolecular disulfide bonds of certain other proteins. Moreparticularly, the invention involves use of thioredoxin and glutaredoxinto reduce gliadins, glutenins, albumins and globulins to improve thecharacteristics of dough and baked goods and create new doughs and toreduce cystine containing proteins such as amylase and trypsininhibitors so as to improve the quality of feed and cereal products.Additionally, the invention involves the isolation of a novel proteinthat inhibits pullulanase and the reduction of that novel protein bythiol redox proteins. The invention further involves the reduction bythioredoxin of 2S albumin proteins characteristic of oil-storing seeds.Also, the invention involves inactivating snake neurotoxins and certaininsect and scorpion venom toxins in vitro and treating the correspondingtoxicities in individuals. The invention also involves using thioredoxinto decrease the allergenicity of food and pollen allergens and toincrease the proteolysis of food and pollen proteins and thedigestibility of food and pollens. The invention also relates to pollenproteins which are reduced by lipoic acid or by reduced thiol-redoxproteins or by thioredoxin in combination with lipoic acid for use inimmunotherapy. The invention further involves use of thiolredox proteinsand lipoic acid to treat and prevent allergies and allergic symptoms.

This invention was made with government support under Grant ContractNos. DCB 8825980 and DMB 88-15980 awarded by the National ScienceFoundation. The United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Chloroplasts contain a ferredoxin/thioredoxin system comprised offerredoxin, ferredoxin-thioredoxin reductase and thioredoxins f and mthat links light to the regulation of enzymes of photosynthesis(Buchanan, B. B. (1991) “Regulation of CO₂ assimilation in oxygenicphotosynthesis: The ferredoxin/thioredoxin system. Perspective on itsdiscovery, present status and future development”, Arch. Biochem.Biophys. 288:1–9; Scheibe, R. (1991), “Redox-modulation of chloroplastenzymes. A common principle for individual control”, Plant Physiol.96:1–3). Several studies have shown that plants also contain a system,analogous to the one established for animals and most microorganisms, inwhich thioredoxin (h-type) is reduced by NADPH and the enzyme,NADP-thioredoxin reductase (NTR) according to the following:

$\begin{matrix}{{NADPH} + H^{+} + {{Thioredoxin}\mspace{20mu}{{\underset{\_}{h}}_{ox}\overset{NTR}{\longrightarrow}{NADP}}} + {{Thioredoxin}\mspace{20mu}{\underset{\_}{h}}_{red}}} & (1)\end{matrix}$(Florencio F. J. et al. (1988), Arch. Biochem. Biophys. 266:496–507;Johnson, T. C. et al. (1987), Plant Physiol. 85:446–451; Suske, G. etal. (1979), Z. Naturforsch. C. 34:214–221). Current evidence suggeststhat the NADP/thioredoxin system is widely distributed in plant tissuesand is housed in the mitochondria, endoplasmic reticulum and cytosol(Bodenstein-Lang, J. et al. (1989), FEBS Lett. 258:22–26; Marcus, F. etal. (1991), Arch. Biochem. Biophys. 287:195–198).

Thioredoxin h is also known to reductively activate cytosolic enzyme ofcarbohydrate metabolism, pyrophosphate fructose-6-P,1-phosphotransferase or PFP (Kiss, F. et al. (1991), Arch. Biochem.Biophys. 287:337–340).

The seed is the only tissue for which the NADP/thioredoxin system hasbeen ascribed physiological activity in plants. Also, thioredoxin h hasbeen shown to reduce thionins in the laboratory (Johnson, T. C. et al.(1987), Plant Physiol. 85:446–451). Thionins are soluble cereal seedproteins, rich in cystine. In the Johnson, et al. investigation, wheatpurothionin was experimentally reduced by NADPH via NADP-thioredoxinreductase (NTR) and thioredoxin h according to Eqs. 2 and 3.

$\begin{matrix}{{NADPH} + {{Thioredoxin}\mspace{20mu}{{\underset{\_}{h}}_{ox}\overset{NTR}{\longrightarrow}{NADP}}} + {{Thioredoxin}\mspace{20mu}{\underset{\_}{h}}_{red}}} & (2)\end{matrix}$Purothionin_(ox)+Thioredoxin h_(red)→Purothionin_(red)+Thioredoxinh_(ox)  (3)

Cereal seeds such as wheat, rye, barley, corn, millet, sorghum and ricecontain four major seed protein groups. These four groups are thealbumins, globulins, gliadins and the glutenins or correspondingproteins. The thionins belong to the albumin group or faction.Presently, wheat and rye are the only two cereals from which gluten ordough has been formed. Gluten is a tenacious elastic and rubbery proteincomplex that gives cohesiveness to dough. Gluten is composed mostly ofthe gliadin and glutenin proteins. It is formed when rye or wheat doughis washed with water. It is the gluten that gives bread dough itselastic type quality. Flour from other major crop cereals barley, corn,sorghum, oat, millet and rice and also from the plant, soybean do notyield a gluten-like network under the conditions used for wheat and rye.

Glutenins and gliadins are cystine containing seed storage proteins andare insoluble. Storage proteins are proteins in the seed which arebroken down during germination and used by the germinating seedling togrow and develop. Prolamines are the storage proteins in grains otherthan wheat that correspond to gliadins while the glutelins are thestorage proteins in grains other than wheat that correspond toglutenins. The wheat storage proteins account for up to 80% of the totalseed protein (Kasarda, D. D. et al. (1976), Adv. Cer. Sci. Tech.1:158–236; and Osborne, T. B. et al. (1893), Amer. Chem. J. 15:392–471).Glutenins and gliadins are considered important in the formation ofdough and therefore the quality of bread. It has been shown from invitro experiments that the solubility of seed storage proteins isincreased on reduction (Shewry, P. R. et al. (1985), Adv. Cer. Sci.Tech. 7:1–83). However, previously, reduction of glutenins and gliadinswas thought to lower dough quality rather than to improve it (Dahle, L.K. et al. (1966), Cereal Chem. 43:682–688). This is probably because thenon-specific reduction with chemical reducing agents caused theweakening of the dough.

The “Straight Dough” and the “Pre-Ferment” methods are two majorconventional methods for the manufacture of dough and subsequent yeastraised bread products.

For the Straight Dough method, all of the flour, water or other liquid,and other dough ingredients which may include, but are not limited toyeast, grains, salt, shortening, sugar, yeast nutrients, doughconditioners, and preservatives are blended to form a dough and aremixed to partial or full development. The resulting dough may be allowedto ferment for a period of time depending upon specific process ordesired end-product characteristics.

The next step in the process is the mechanical or manual division of thedough into appropriate size pieces of sufficient weight to ensureachieving the targeted net weight after baking, cooling, and slicing.The dough pieces are often then rounded and allowed to rest(Intermediate Proof) for varying lengths of time. This allows the doughto “relax” prior to sheeting and molding preparations. The timegenerally ranges from 5–15 minutes, but may vary considerably dependingon specific processing requirements and formulations. The dough piecesare then mechanically or manually formed into an appropriate shape arethen usually given a final “proof” prior to baking. The dough pieces arethen baked at various times, temperatures, and steam conditions in orderto achieve the desired end product.

In the Pre-Ferment method, yeast is combined with other ingredients andallowed to ferment for varying lengths of time prior to final mixing ofthe bread or roll dough. Baker's terms for these systems include “WaterBrew”, “Liquid Ferment”, “Liquid Sponge”, and “Sponge/Dough”. Apercentage of flour ranging from 0–100% is combined with the otheringredients which may include but are not limited to water, yeast, yeastnutrients and dough conditioners and allowed to ferment under controlledor ambient conditions for a period of time. Typical times range from 1–5hours. The ferment may then be used as is, or chilled and stored in bulktanks or troughs for later use. The remaining ingredients are added(flour, characterizing ingredients, additional additives, additionalwater, etc.) and the dough is mixed to partial or full development.

The dough is then allowed to ferment for varying time periods.Typically, as some fermentation has taken place prior to the addition ofthe remaining ingredients, the time required is minimal (i.e., 10–20min), however, variations are seen depending upon equipment and producttype. Following the second fermentation step, the dough is then treatedas in the Straight Dough Method.

As used herein the term “dough mixture” describes a mixture thatminimally comprises a flour or meal and a liquid, such as milk or water.

As used herein the term “dough” describes an elastic, pliable proteinnetwork mixture that minimally comprises a flour, or meal and a liquid,such as milk or water.

As used herein the term “dough ingredient” may include, but is notexclusive of, any of the following ingredients: flour, water or otherliquid, grain, yeast, sponge, salt, shortening, sugar, yeast nutrients,dough conditioners and preservatives.

As used herein, the term “baked good” includes but is not exclusive ofall bread types, including yeast-leavened and chemically-leavened andwhite and variety breads and rolls, english muffins, cakes and cookies,confectionery coatings, crackers, doughnuts and other sweet pastrygoods, pie and pizza crusts, pretzels, pita and other flat breads,tortillas, pasta products, and refrigerated and frozen dough products.

While thioredoxin has been used to reduce albumins in flour, thiol redoxproteins have not been used to reduce glutenins and gliadins nor otherwater insoluble storage proteins, nor to improve the quality of doughand baked goods. Thiol redox proteins have also not been used to improvethe quality of gluten thereby enhancing its value nor to prepare doughfrom crop cereals such as barley, corn, sorghum, oat, millet and rice orfrom soybean flour.

Many cereal seeds also contain proteins that have been shown to act asinhibitors of enzymes from foreign sources. It has been suggested thatthese enzyme inhibitors may afford protection against certaindeleterious organisms (Garcia-Olmedo, F. et al. (1987), Oxford Surveysof Plant Molecular and Cell Biology 4:275–335; Birk, Y. (1976), Meth.Enzymol. 45:695–739, and Laskowski, M., Jr. et al. (1980), Ann. Reo.Biochem. 49:593–626). Two such type enzyme inhibitors are amylaseinhibitors and trypsin inhibitors. Furthermore, there is evidence that abarley protein inhibitor (not tested in this study) inhibits anα-amylase from the same source (Weselake, R. J. et al. (1983), PlantPhysiol. 72:809–812). Unfortunately, the inhibitor protein often causesundesirable effects in certain food products. The trypsin inhibitors insoybeans, notably the Kunitz trypsin inhibitor (KTI) and Bowman-Birktrypsin inhibitor (BBTI) proteins, must first be inactivated before anysoybean product can be ingested by humans or domestic animals. It isknown that these two inhibitor proteins become ineffective as trypsininhibitors when reduced chemically by sodium borohydride (Birk, Y.(1985), Int. J. Peptide Protein Res. 25:113–131, and Birk, Y. (1976),Meth. Enzymol. 45:695–739). These inhibitors like other proteins thatinhibit proteases contain intramoelcular disulfides and are usuallystable to inactivation by heat and proteolysis (Birk (1976), supra.;Garcia-Olmedo et al. (1987), supra., and Ryan (1980). Currently, tominimize the adverse effects caused by the inhibitors these soybeantrypsin inhibitors and other trypsin inhibitors in animal and human foodproducts are being treated by exposing the food to high temperatures.The heat treatment, however, does not fully eliminate inhibitoractivity. Further, this process is not only expensive but it alsodestroys many of the other proteins which have important nutritionalvalue. For example, while 30 min at 120° C. leads to completeinactivation of the BBTI of soy flour, about 20% of the original KTIactivity remains (Friedman et al., 1991). The prolonged or highertemperature treatments required for full inactivation of inhibitorsresults in destruction of amino acids such as cystine, arginine, andlysine (Chae et al., 1984; Skrede and Krogdahl, 1985).

There are also several industrial processes which require α-amylaseactivity. One example is the malting of barley which requires activeα-amylase. Inactivation of inhibitors such as the barleyamylase/subtilisin (asi) inhibitor and its equivalent in other cerealsby thiol redox protein reduction would enable α-amylases to become fullyactive sooner than with present procedures, thereby shortening time formalting or similar processes.

Thiol redox proteins have also not previously been used to inactivatetrypsin or amylase inhibitor proteins. The reduction of trypsininhibitors such as the Kunitz and Bowman-Birk inhibitor proteinsdecreases their inhibitory effects (Birk, Y. (1985), Int. J. PeptideProtein Res. 25:113–131). A thiol redox protein linked reduction of theinhibitors in soybean products designed for consumption by humans anddomestic animals would require no heat or lower heat than is presentlyrequired for protein denaturization, thereby cutting the costs ofdenaturation and improving the quality of the soy protein. Also aphysiological reductant, a so-called clean additive (i.e., an additivefree from ingredients viewed as “harmful chemicals”) is highly desirablesince the food industry is searching for alternatives to chemicaladditives. Further the ability to selectively reduce the major wheat andseed storage proteins which are important for flour quality (e.g., thegliadins and the glutenins) in a controlled manner by a physiologicalreductant such as a thiol redox protein would be useful in the bakingindustry for improving the characteristics of the doughs from wheat andrye and for creating doughs from other grain flours such as cerealflours or from cassava or soybean flour.

The family of 2S albumin proteins characteristic of oil-storing seedssuch as castor bean and Brazil nut (Kreis et al. 1989; Youle and Huang,1981) which are housed within protein bodines in the seed endosperm orcotyledons (Ashton et al. 1976; Weber et al. 1980), typically consist ofdissimilar subunits connected by two intermolecular disculfide bonds—onesubunit of 7 to 9 kDa and the other of 3 to 4 kDa. The large subunitcontains two intramolecular disculfide groups, the small subunitcontains none. The intramolecular disculfides of the 2S large subunitshow homology with those of the soybean Bowman-Birk inhibitor (Kreis etal. 1989) but nothing is known of the ability of 2S proteins to undergoreduction under physiological conditions.

These 2S albumin proteins are rich in methionine. Recently transgenicsoybeans which produce Brazil nut 2S protein have been generated.Reduction of the 2S protein in such soybeans could enhance theintegration of the soy proteins into a dough network resulting in asoybread rich in methionine. In addition, these 2S proteins are oftenallergens. Reduction of the 2S protein would result in the cessation ofits allergic activity. Pullulanase (“debranching enzyme”) is an enzymethat breaks down the starch of the endosperm of cereal seeds byhydrolytically cleaving α-1,6 bonds. Pullulanase is an enzymefundamental to the brewing and baking industries. Pullulanase isrequired to break down starch in malting and in certain bakingprocedures carried out in the absence of added sugars or othercarbohydrates. Obtaining adequate pullulanase activity is a problemespecially in the malting industry. It has been known for some time thatdithiothreitol (DTT, a chemical reductant for thioredoxin) activatespullulanase of cereal preparations (e.g., barley, oat and rice flours).A method for adequately activating or increasing the activity ofpullulanase with a physologically acceptable system, could lead to morerapid malting methods and, owing to increased sugar availability, toalcoholic beverages such as beers with enhanced alcoholic content.

Death and permanent injury resulting from snake bites are seriousproblems in many African, Asian and South American countries and also amajor concern in several southern and western areas of the UnitedStates. Venoms from snakes are characterized by active proteincomponents (generally several) that contain disulfide (S—S) bridgeslocated in intramolecular (intrachain) cystines and in some cases inintermolecular (interchain) cystines. The position of the cystine withina given toxin group is highly conserved. The importance ofintramolecular S—S groups to toxicity is evident from reports showingthat reduction of these groups leads to a loss of toxicity in mice(Yang, C. C. (1967) Biochim. Biophys. Acta. 133:346–355; Howard, B. D.et al. (1977) Biochemistry 16:122–125). The neurotoxins of snake venomare proteins that alter the release of neurotransmitter from motor nerveterminals and can be presynaptic or postsynaptic. Common symptomsobserved in individuals suffering from snake venom neurotoxicity includeswelling, edema and pain, fainting or dizziness, tingling or numbing ofaffected part, convulsions, muscle contractions, renal failure, inaddition to long-term necrosis and general weakening of the individual,etc.

The presynaptic neurotoxins are classified into two groups. The firstgroup, the β-neurotoxins, include three different classes of proteins,each having a phospholipase A₂ component that shows a high degree ofconservation. The proteins responsible for the phospholipase A₂ activityhave from 6 to 7 disulfide bridges. Members of the β-neurotoxin groupare either single chain (e.g., caudotoxin, notexin and agkistrodotoxin)or multichain (e.g., crotoxin, ceruleotoxin and Vipera toxin).β-bungarotoxin, which is made up of two subunits, constitutes a thirdgroup. One of these subunits is homologous to the Kunitz-type proteinaseinhibitor from mammalian pancreas. The multichain β-neurotoxins havetheir protein components linked ionically whereas the two subunits ofβ-bungarotoxin are linked covalently by an intermolecular disulfide. TheB chain subunit of β-bungarotoxin, which is also homologous to theKunitz-type proteinase inhibitor from mammalian pancreas, has 3disulfide bonds.

The second presynaptic toxin group, the facilitatory neurotoxins, isdevoid of enzymatic activity and has two subgroups. The first subgroup,the dendrotoxins, has a single polypeptide sequence of 57 to 60 aminoacids that is homologous with Kunitz-type trypsin inhibitors frommammalian pancreas and blocks voltage sensitive potassium channels. Thesecond subgroup, such as the fasciculins (e.g., fasciculin 1 andfasiculin 2) are cholinesterase inhibitors and have not been otherwiseextensively studied.

The postsynaptic neurotoxins are classified either as long or shortneurotoxins. Each type contains S—S groups, but the peptide is uniqueand does not resemble either phospholipase A₂ or the Kunitz orKunitz-type inhibitor protein. The short neurotoxins (e.g., erabutoxin aand erabutoxin b) are 60 to 62 amino acid residues long with 4intramolecular disulfide bonds. The long neurotoxins (e.g.,α-bungarotoxin and α-cobratoxin) contain from 65 to 74 residues and 5intramolecular disulfide bonds. Another type of toxins, the cytotoxins,acts postsynaptically but its mode of toxicity is ill defined. Thesecytotoxins show obscure pharmacological effects, e.g., hemolysis,cytolysis and muscle depolarization. They are less toxic than theneurotoxins. The cytotoxins usually contain 60 amino acids and have 4intramolecular disulfide bonds. The snake venom neurotoxins all havemultiple intramolecular disulfide bonds.

The current snake antitoxins used to treat poisonous snake bitesfollowing first aid treatment in individuals primarily involveintravenous injection of antivenom prepared in horses. Although it isnot known how long after envenomation the antivenom can be administeredand be effective, its use is recommended up to 24 hours. Antivenomtreatment is generally accompanied by administration of intravenousfluids such as plasma, albumin, platelets or specific clotting factors.In addition, supporting medicines are often given, for example,antibiotics, antihistamines, antitetanus agents, analgesics andsedatives. In some cases, general treatment measures are taken tominimize shock, renal failure and respitory failure. Other thanadministering calcium-EDTA in the vicinity of the bite and excising thewound area, there are no known means of localized treatment that resultin toxin neutralization and prevention of toxic uptake into the bloodstream. Even these localized treatments are of questionable significanceand are usually reserved for individuals sensitive to horse serum(Russell, F. E. (1983) Snake Venom Poisoning, Schollum International,Inc. Great Neck, N.Y.).

The term “individual” as defined herein refers to an animal or a human.

Most of the antivenoms in current use are problematic in that they canproduce harmful side effects in addition to allergic reactions inpatients sensitive to horse serum (up to 5% of the patients).Nonallergic reactions include pyrogenic shock, and complement depletion(Chippaur, J.-P. et al. (1991) Reptile Venoms and Toxins, A. T. Tu, ed.,Marcel Dekker, Inc., pp. 529–555).

It has been shown that thioredoxin, in the presence of NADPH andthioredoxin reductase reduces the bacterial neurotoxins tetanus andbotulinum A in vitro (Schiavo, G. et al. (1990) Infection and Immunity58:4136–4141; Kistner, A. et al. (1992) Naunyn-Schmiedeberg's ArchPharmacol 345:227–234). Thioredoxin was effective in reducing theinterchain disulfide link of tetanus toxin and such reduced tetanustoxin was no longer neurotoxic (Schiavo et al., supra.). However,reduction of the interchain disulfide of botulinum A toxin bythioredoxin was reported to be much more sluggish (Kistner et al.,supra.). In contrast to the snake neurotoxin studied in the course ofthis invention, the tetanus research group (Schiavo et al., supra.)found no evidence in the work done with the tetanus toxin that reducedthioredoxin reduced toxin intrachain disulfide bonds. There was also noevidence that thioredoxin reduced intrachain disulfides in the work donewith botulinum A. The tetanus and botulinum A toxins are significantlydifferent proteins from the snake neurotoxins in that the latter (1)have a low molecular weight; (2) are rich in intramolecular disulfidebonds; (3) are resistant to trypsin and other animal proteases; (4) areactive without enzymatic modification, e.g., proteolytic cleavage; (5)in many cases show homology to animal proteins, such as phospholipase A₂and Kunitz-type proteases; (6) in most cases lack intermoleculardisulfide bonds, and (7) are stable to agents such as heat andproteases.

Reductive inactivation of snake toxins in vitro by incubation with 1%β-mercaptoethanol for 6 hours and incubation with 8M urea plus 300 mMβ-mercaptoethanol has been reported in the literature (Howard, B. D. etal. (1977) Biochemistry 16:122–125; Yang, C. C. (1967) Biochim. Biophys.Acta. 133:346–355). These conditions, however, are far fromphysiological. As defined herein the term “inactivation” with respect toa toxin protein means that the toxin is no longer biologically active invitro, in that the toxin is unable to link to a receptor. Also as usedherein, “detoxification” is an extension of the term “inactivation” andmeans that the toxin has been neutralized in an individual as determinedby animal toxicity tests.

Bee venom is a complex mixture with at least 40 individual components,that include major components as melittin and phospholipase A₂,representing respectively 50% and 12% of the total weight of the venom,and minor components such as small proteins and peptides, enzymes,amines, and amino acids.

Melittin is a polypeptide consisting of 26 amino acids with a molecularweight of 2840. It does not contain a disulfide bridge. Owing to itshigh affinity for the lipid-water interphase, the protein permeates thephospholipid bilayer of the cell membranes, disturbing its organizedstructure. Melittin is not by itself a toxin but it alters the structureof membranes and thereby increases the hydrolitic activity ofphospholipase A₂, the other major component and the major allergenpresent in the venom.

Bee venom phospholipase A₂ is a single polypeptide chain of 128 aminoacids, is cross-linked by four disulfide bridges, and containscarbohydrate. The main toxic effect of the bee venom is due to thestrong hydrolytic activity of phospholipase A₂ achieved in associationwith melittin.

The other toxic proteins in bee venom have a low molecular weight andcontain at least two disulfide bridges that seem to play an importantstructural role. Included are a protease inhibitor (63–65 amino acids),MCD or 401-peptide (22 amino acids) and apamin (18 amino acids).

Although there are thousands of species of bees, only the honey bee,Apis mellifera, is a significant cause of allergic reactions. Theresponse ranges from local discomfort to systemic reactions such asshock, hypotension, dyspnea, loss of consciousness, wheezing and/orchest tightness that can result in death. The only treatment that isuseed in these cases is the injection of epinephrine.

The treatment of bee stings is important not only for individuals withallergic reactions. The “killer” or Africanized bee, a variety of honeybee is much more agressive than European honey bees and represents adanger in both South and North America. While the lethality of the venomfrom the Africanized and European bees appears to be the same(Schumacher, M. I. et al. (1989) Nature 337:413), the behaviour patternof the hive is completely different. It was reported that Africanizedbees respond to colony disturbance more quickly, in greater numbers andwith more stinging (Collins, A. M. et al. (1982) Science 218:72–74). Amass attack by Africanized bees may produce thousands of stings on oneindividual and cause death. The “killer” bees appeared as a result ofthe interbreeding between the African bee (Apis mellifera scutellata)and the European bee (Apis mellifera mellifera). African bees wereintroduced in 1956 into Brazil with the aim of improving honeyproduction being a more tropically adapted bee. Africanized bees havemoved from South America to North America, and they have been reportedin Texas and Florida.

In some areas of the world such as Mexico, Brazil, North Africa and theMiddle East, scorpions present a life hazard to humans. However, onlythe scorpions of family Buthidae (genera, Androctonus, Buthus,Centruroides, Lejurus and Tityus) are toxic for humans. The chemicalcomposition of the scorpion venom is not as complex as snake or beevenom. Scorpion venom contains mucopolysaccharides, small amounts ofhyaluronidase and phospholipase, low molecular-weight molecules,protease inhibitors, histamine releasers and neurotoxins, such asserotonin. The neurotoxins affect voltage-sensitive ionic channels inthe neuromuscular junction. The neurotoxins are basic polypeptides withthree to four disulflde bridges and can be classified in two groups:peptides with from 61 to 70 amino acids, that block sodium channel, andpeptides with from 36 to 39 amino acids, that block potassium channel.The reduction of disulfide bridges on the neurotoxins bynonphysiological reductants such as DTT or β-mercaptoethanol (Watt, D.D. et al. (1972) Toxicon 10:173–181) lead to loss of their toxicity.

Symptoms of animals stung by poisonous scorpions inclurehyperexcitability, dyspnea, convulsions, paralysis and death. Atpresent, antivenin is the only antidote for scorpion stings. Theavailability of the venom is a major problem in the production ofantivenin. Unlike snake venom, scorpion venom is very difficult tocollect, because the yield of venom per specimen is limited and in somecases the storage of dried venom leads to modification of its toxicity.An additional problem in the production of antivenins is that theneurotoxins are very poor antigens.

The reductive inactivation of snake, bee and scorpion toxins underphysiological conditions has never been reported nor has it beensuggested that the thiol redox agents, such as reduced lipoic acid, DTT,or reduced thioredoxin could act as an antidote to these venoms in anindividual.

Food allergies also represent a long-standing problem important bothnationally and internationally. Up to 5% of children under age 12 and 1%of adults are clinically affected in the U.S. population (AdverseReactions to Foods—AAAI and NIAD Report, 1984, NIH Pub. No. 84–2442, pp.2, 3). In some countries, the figures are higher, and, throughout theworld, the problem is considered to be increasing, especially in infants(T. Matsuda and R. Nakamura 1993 Molecular structure and immunologicalproperties of Food Allergens, Trends in Food Science & Technology 4,289–293). The problem extends to a wide range of foods. Food allergiesin general have recently achieved an increased profile as a result ofthe concern about transgenic foods.

Milk represents a significant problem, especially in infants. Wheat andsoy allergies are of growing importance as new populations adopt thesefoods and are of increased concern in pet (especially dog) foods. Beef,rice and egg also cause serious allergies in many individuals and againare of significant concern with respect to pet food.

Many of the major allergenic proteins in the above mentioned foods haveintramolecular disulfide (S—S) bonds but so far two treatments have beenapplied commercially to minimize food allergies: (1) heat, and (2)enzymatic proteolysis. In both cases, success has been only partial.While lowering allergenicity, heat treatment has not eliminated theproblem, even in the best of cases, because the responsible proteins aretypically heat stable. Moreover, heat lowers product quality bydestroying nutritionally important amino acids such as lysine, cysteineand arginine. Enzymatic proteolysis is more successful in reducingallergenicity, but desirable food properties such as flavor are usuallylost and treatment is costly. Therefore a physiologically safe systemthat would bring about a decrease in or loss of allergenicity whenapplied to allergenic foods without a resulting loss in flavor andnutrition would be extremely valuable.

Certain major pollen allergens are known to be disulfide proteins thatare highly resistant to temperature. Two pollen proteins are describedas major allergens in ragweed pollen. One is a small protein of 5 kDa,Amb a V, containing four disulfide bridges (Goodfriend, L. et al.(1985), “Ra5G, a homologue of Ra5 in giant ragweed pollen:isolation,HLA-DR-associated activity and amino acid sequence”, Mol. Immunol.22:899–906; Metzler, W. J. et al. (1992), “Determination of thethree-dimensional solution structure of ragweed allergen Amb t V bynuclear magnetic resonance spectroscopy” Biochemistry 31:5117–5127;Mole, L. E., et al. (1975), “The amino acid sequence of ragweed pollenallergen Ra5” Biochemistry 14:1216–1220; Metzler, W. J., et al. (1992),“Proton resonance assignments and three-dimensional solution structureof the ragweed allergen Amb a V by nuclear magnetic resonancespectroscopy” Biochemistry 31:8697–8705). This protein is considered tobe homologous in both the short and giant ragweed species. The shortragweed protein which is designated Amb a V and the giant ragweed whichis now designated Amb t V, both previously called Ra 5, exhibit a 45%sequence similarity.

The other major allergen represents a family of 41 kDa proteins, namedAmb a 1.1, Amb a 1.2, Amb a 1.3 and Amb a 1.4. While no disulfidebridges have been described, these proteins contain multiple cysteines(Rafnar, T. et al. (1991), “Cloning of Amb a I (antigen E), the majorallergen family of short ragweed pollen” J. Biol. Chem. 266:1229–1236;Griffith, I. J. et al. (1991), “Sequence polymorphism of Amb a I and Amba II, the major allergens in Ambrosia artemisiifolia (short ragweed)”Int. Arch. Allergy Appl. Immunol. 96:296–304). Yet other known allergensare disulfide proteins such as the western ragweed, Amb P 5-A and -B,each 8.5 kDa with three disulfide bridges (Ghosh, B. et al. (1994),“Immunologic and molecular characterization of Amb p V allergens fromAmbrosia psilostachya (western Ragweed) pollen” J. Immunol.152:2882–2889) and a short ragweed 11.4. kDa plastocyanin like protein,caUed Ra 3, with one disulfide bridge (Klapper, D. G. et al. (1980),“Amino acid sequence of ragweed allergen Ra3” Biochemistry19:5729–5734).

The 5 kDa Amb V ragweed pollen proteins have a well-defined structureand the positions of the four intrachain disulfide bonds are preciselyknown (Metzler, W. J. et al. (1992) Biochemistry 31:5117–5127 and8697–8705). Previous work has shown that, when reduced under denaturingconditions by chemical agents (urea plus either dithiothreitol orβ-mercaptoethanol), the immune response shifts from IgE (allergic) to anIgG (defense) because IgG production is enhanced (Zhu, X. et al. (1995),“T cell epitope mapping of ragweed pollen allergen Ambrosiaartemisiifolia (Amb a 5) and Ambrosia trifida (Amb t 5) and the role offree sulfhydryl groups in T cell recognition” J. Immunol. 155:5064–73).

Pollen allergies are currently being treated by conventionalimmunotherapy with undenatured pollen extract. However, such treatment,especially in children, carries a certain risk of anaphylactic reactionswhich are potentially lethal. Consequently, there is a need for anattenuated pollen protein or pollen extract for use in immunotherapythat would reduce or eliminate the possibility of anaphylacticreactions. There is also a need for a physiologically safe system thatcould determine whether or not an allergen for a particular individualis a disulfide protein. Further, eye drops, nose sprays, aerosols, ordispersants for vaporizers or humidifiers that would alleviate allergysymptoms but also produce less side effects than the currently availableproducts would be extremely valuable.

SUMMARY OF THE INVENTION

It is an object herein to provide a method for reducing a non thionincystine containing protein.

It is a second object herein to provide methods utilizing a thiol redoxprotein alone or in combination with a reductant or reduction system toreduce glutenins or gliadins present in flour or seeds.

It is also an object herein to provide methods using a thiol redoxprotein alone or in combination with a reductant or reduction system toimprove dough strength and baked goods characteristics such as bettercrumb quality, softness of the baked good and higher loaf volume.

It is a further object herein to provide formulations containing a thiolredox protein useful in practicing such methods.

Still a further object herein is to provide a method for producing adough from rice, corn, soybean, barley, oat, cassava, sorghum or milletflour.

Yet another object is to provide a method for producing an improvedgluten or for producing a gluten-like product from cereal grains otherthan wheat and rye.

It is further an object herein to provide a method of reducing an enzymeinhibitor protein having disulfide bonds.

Still another object herein is to provide yeast cells geneticallyengineered to express or overexpress thioredoxin.

Still yet another object herein is to provide yeast cells geneticallyengineered to express or overexpress NADP-thioredoxin reductase.

Still yet a further object herein is to provide a method for improvingthe quality of dough or a baked good using such genetically engineeredyeast cells.

Yet still another object herein is to provide a method of reducing theintramolecular disulfide bonds of a non-thionin, non chloroplast proteincontaining more than one intramolecular cystine comprising adding athiol redox protein to a liquid or substance containing the cystinescontaining protein, reducing the thiol redox protein and reducing thecystines containing protein by means of the thiol redox protein.

Another object herein is to provide an isolated pullulanase inhibitorprotein having disulfide bonds and a molecular weight of between 8 to 15kDa.

Still another object herein is to provide a method of increasing theactivity of pullulanase derived from barley or wheat endospermcomprising adding thioredoxin to a liquid or substance containing thepullulanase and reducing the thioredoxin thereby increasing thepullulanase activity.

Still another object herein is to provide a method of reducing an animalvenom toxic protein having one or more intramolecular cystinescomprising contacting the cystine containing protein with an amount of athiol redox (SH) agent effective for reducing the protein, andmaintaining the contact for a time sufficient to reduce one or moredisulfide bridges of the one or more intramolecular cystines therebyreducing the neurotoxin protein. The thiol redox (SH) agent may be areduced thioredoxin, reduced lipoic acid in the presence of athioredoxin, DTT or DTT in the presence of a thioredoxin and the snakeneurotoxin protein may be a presynaptic or postsynaptic neurotoxin.

Still a further object of the invention is to provide a compositioncomprising a snake neurotoxin protein and a thiol redox (SH) agent.

Still yet another object of the invention is to provide a method ofreducing an animal venom toxic protein having one or more intramolecularcystines comprising contacting the protein with amounts ofNADP-thioredoxin reductase, NADPH or an NADPH generator system and athioredoxin effective for reducing the protein, and maintaining thecontact for a time sufficient to reduce one or more disulfide bridges ofthe one or more intramolecular cystines thereby reducing the protein.

Yet another object herein is to provide a method of inactivating, invitro, a snake neurotoxin having one or more intramolecular cystinescomprising adding a thiol redox (SH) agent to a liquid containing thetoxin wherein the amount of the agent is effective for reducing thetoxin.

Yet a further object herein is to provide a method of treating venomtoxicity in an individual comprising administering, to an individualsuffering from venom toxicity, amounts of a thiol redox (SH) agenteffective for reducing or alleviating the venom toxicity.

In accordance with the objects of the invention, methods are providedfor improving dough characteristics comprising the steps of mixing athiol redox protein with dough ingredients to form a dough and bakingsaid dough.

Also, in accordance with the objects of the invention, a method isprovided for inactivating an enzyme inhibitor protein in a grain foodproduct comprising the steps of mixing a thiol redox protein with theseed product, reducing the thiol redox protein by a reductant orreduction system and reducing the enzyme inhibitor by the reduced thiolredox protein, the reduction of the enzyme inhibitor inactivating theenzyme inhibitor.

The thiol redox proteins in use herein can include thioredoxin andglutaredoxin. The thioredoxin includes but is not exclusive of E. colithioredoxin, thioredoxin h, f and m and animal thioredoxins. A reductantof thioredoxin used herein can include lipoic acid or a reduction systemsuch as NADPH in combination with NADP thioredoxin reductase (NTR). Thereductant of glutaredoxin can include reduced glutathione in conjunctionwith the reduction system NADPH and glutathione reductase. NADPH can bereplaced with an NADPH generator or generator composition such as oneconsisting of glucose 6-phosphate, NADP and glucose 6-phosphatedehydrogenase from a source such as yeast. The NADPH generator is addedtogether with thioredoxin and NADP-thioredoxin reductase at the start ofthe dough making process.

It should be noted that the invention can also be practiced withcysteine containing proteins. The cysteines can first be oxidized andthen reduced via thiol redox protein.

Further in accordance with the objects of the invention, a method isprovided for decreasing the allergenicity of an allergenic food proteincomprising the steps of contacting the protein with an amount ofthioredoxin, NTR and NADPH or an amount of DTT in the presence ofthioredoxin effective for decreasing the allergenicity of the proteinand administering the contacted protein in step (a) to an animal,thereby decreasing the allergenic symptoms in said animal that wouldotherwise occur if the animal received the untreated protein.

Another object of the invention is to provide a hypo-allergenicingestible food. The food was made hypo-allergenic by prior treatmentwith thioredoxin in the presence of NTR and NADPH. The food can be beef,milk, soy, egg, rice or wheat.

A further object of the invention is to provide a method for improvingthe proteolysis and therefore the digestibility of food and allergenproteins and consequently to also provide more digestible foods, many ofwhich are allergenic. The foods and allergens are made more susceptibleto proteolysis and more digestible by prior treatment with thioredoxinin the presence of reductants of thioredoxin such as those describedabove.

Appropriate foods include soy, nuts, milk, whey, beef, egg, bread, otherwheat products, and other grain products.

Still another object of the invention is to provide a method fordecreasing the allergenicity of an allergenic pollen protein comprisingthe steps of contacting the protein with an amount of reducedthioredoxin effective for decreasing the allergenicity of the proteinand administering the thioredoxin reduced protein to an animal inimmunotherapeutic doses thereby decreasing the allergenic symptoms ofsaid animal that would otherwise occur if the animal was exposed to theuntreated protein.

Yet another object of the invention is to provide a hypo-allergenicpollen or pollen protein with reduced disulfide bonds forinmmunotherapy.

A further object of this invention is to provide a method fordetermining whether or not an allergen for a particular individual is adisulfide protein comprising administering an allergy test to saidindividual to identify said allergen, treating said identified allergenprotein in vitro with reduced thioredoxin and analyzing said treatedallergen protein for disulfide bond reduction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the mitigation of a skin test response tosoy allergen by treating soy allergenic extract with reducedthioredoxin.

FIG. 2 is a bar graph showing the mitigation of a skin test response tomilk allergen by treating milk allergenic extract with reducedthioredoxin.

FIG. 3 is a bar graph showing the mitigation of a skin test response towheat allergen by treating wheat allergenic extract with reducedthioredoxin.

FIG. 4 is a bar graph showing the mitigation of a skin test response tobeef allergen by treating beef allergenic extract with reducedthioredoxin at room temperature.

FIG. 5 is a bar graph showing the mitigation of a skin test response tobeef allergen by treating beef allergenic extract with reducedthioredoxin at 37° C.

FIG. 6 is a bar graph showing the mitigation of a gastrointestinalallergenic response to soy and wheat by treating diets with reducedthioredoxin.

FIG. 7 is a photograph of an SDS polyacrylamide electrophoretic gelshowing the extent of thioredoxin-linked and glutathione-linkedreduction by means of fluorescence and protein staining.

FIG. 8 is a drawing of the tertiary structure of oxidized bovineβ-lactoglobulin showing the disulfide bridges and free sulflhydryl.

FIG. 9 is a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of thioredoxin-linked reductionon pepsin digestion of bovine β-lactoglobulin.

FIG. 10A represents a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of time on the digestion ofuntreated milk.

FIG. 10B represents a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of time on the digestion of milkfollowing thioredoxin-linked reduction that occurred at 55° C.

FIG. 10C represents a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of time on the digestion of milkfollowing thioredoxin-linked reduction that occurred at 4° C.

FIG. 11A is a bar graph showing the thioredoxin-linked mitigation of askin test response in a dog highly sensitive to milk.

FIG. 11B is a bar graph showing the thioredoxin-linked mitigation of askin test response in a dog mildly sensitive to milk.

FIG. 12A is a bar graph showing the effect of thioredoxin-linkedreduction and digestibility on the allergenicity of β-lactoglobulin.

FIG. 12B is a bar graph showing the effect of thioredoxin-linkedreduction and digestibility on the allergenicity of milk.

FIG. 13 is a drawing of the tertiary structure of oxidized bovineβ-lactoglobulin showing the mouse antibody (MAb) epitopes.

FIG. 14 is a graph of a computer generated molecular model of thetertiary structure of bovine β-lactoglobulin showing predictedmonoclonal antibody epitope changes after cystine mutagenesis.

FIG. 15 is a photograph of an SDS polyacrylamide electrophoretic gelshowing the extent of thioredoxin-linked and glutathione-linkedreduction of ragweed pollen allergens by means of fluorescence.

FIG. 16A is a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of time on the digestion ofuntreated ragweed allergen Amb t V.

FIG. 16B is a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of time on the digestion ofragweed allergen Amb t V following thioredoxin-linked reduction.

FIG. 17A is a bar graph showing the effect of thioredoxin-linkedreduction and pepsin on the allergenicity of giant ragweed pollenextract in dogs that are sensitive to disulfide bond containingproteins.

FIG. 17B is a bar graph showing the effect of thioredoxin-linkedreduction and pepsin digestibility on the allergenicity of giant ragweedpollen extract in dogs less sensitive to disulfide bond containingproteins.

FIG. 18A is a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of time on the digestion ofragweed pollen following thioredoxin-linked reduction that occurred at4° C.

FIG. 18B is a photograph of a protein stained SDS polyacrylamideelectrophoretic gel showing the effect of time on the digestion ofragweed pollen following thioredoxin-linked reduction at 37° C. and 55°C.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this detailed description, the following definitionsand abbreviations apply:

CM certain bread wheat α-amylase inhibitors DSG certain α-amylaseinhibitors isolated from durum wheat DTNB 2′5′-dithiobis (2-nitrobenzoicacid) NTR NADP-thioredoxin reductase mBBr monobromobimane NADP-MDHNADP-malate dehydrogenase FBPase fructose-1,6-bisphosphatase SDS sodiumdodecyl sulfate DTT dithiothreitol Cereal millet, wheat, oat, barley,rice, sorghum, or rye BBTI Bowman-Birk soybean trypsin inhibitor KTIKunitz soybean trypsin inhibitor PAGE polyacrylamide gel electrophoresisTCA trichloroacetic acid

ENZYME INHIBITOR PROTEIN EXPERIMENTS Starting Materials

Materials

Seeds of bread wheat Triticum aestivum L, cv. Talent) and durum wheat(Triticum durum. Desf., cv. Mondur) were obtained from laboratorystocks.

Reagents

Chemicals and fine chemicals for enzyme assays and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis were purchased fromSigma Chemical Co. and BioRad Laboratories, respectively.Monobromobimane (mBBr, tradename Thiolite) was purchased fromCalbiochem. Other chemicals were obtained from commercial sources andwere of the highest quality available.

Enzymes

Thioredoxin and NTR from E. coli ware purchased from AmericanDiagnostics, Inc. and were also isolated from cells transformed tooverexpress each protein. The thioredoxin strain containing therecombinant plasmid, pFP1, was kindly provided by Dr. J.-P. Jacquot (dela Motte-Guery, F. et al. (1991) Eur. J. Biochem. 196:287–294). The NTRstrain containing the recombinant plasmid, pPMR21, was kindly providedby Drs. Marjorie Russel and Peter Model (Russel, M. et al. (1988) J.Biol. Chem. 263:9015–9019). The Isolation procedure used for theseproteins was as described in those studies with the following changes:cells were broken in a Ribi cell fractionator at 25,000 psi and NTR waspurified as described by Florencio et al. (Fiorencio. F. J. et al.(1988) Arch. Biochem. Biophys. 266:496–507) without the red agarosestep. Thioredoxin and NTR from Saccharomyces cerevisiae (baker's yeasttype 1) were isolated by the procedure developed by Florencio et al. forspinach leaves with the following changes: suspended cells [1 partcells:5 parts buffer (w/v)], were broken in a Ribi cell fractionator at40,000 psi with three passes.

Thioredoxin h and NTR were isolated from wheat germ by the proceduredeveloped for spinach leaves (Florencio, F. J. et al. (1988), Arch.Biochem. Biophys. 266:496–507). NADP-malate dehydrogenase (NADP-MDH) andfructose-1,6-bisphosphatase (FBPase) were purified from leaves of corn(Jacquot, J.-P. et al. (1981), Plant Physiol. 68:300–304) and spinach(Crawford, N. A. et al. (1989), Arch. Biochein. Biophys. 271:223–239)respectively. E. coli glutaredoxin and calf thymus thioredoxin wereobtained from Professor A. Holmgren.

α-Amylase and Trypsin Inhibitors

CM-1 protein was isolated from the albumin-globulin fraction of breadwheat flour as described previously (Kobrehel, K. et al. (1991), CerealChem. 68:1–6). A published procedure was also used for the isolation ofDSG proteins (DSG-1 and DSG-2) from the glutenin fraction of durum wheat(Kobrehel, K. et al. (1989), J. Sci. Food Agric. 48:441–452). The CM-1,DSG-1 and DSG-2 proteins were homogeneous in SDS-polyacrylamide gelelectrophoresis. Trypsin inhibitors were purchased from Sigma ChemicalCo., except for the one from corn kernel which was from Fluca. In allcases, the commercial preparations showed a single protein componentwhich migrated as expected in SDS-PAGE (Coomassie Blue stain), but incertain preparations, the band was not sharp.

Other Proteins

Purothionin α from bread wheat and purothionins α-1 and β from durumwheat were kind gifts from Drs. D. D. Kasarda and B. L. Jones,respectively. The purothionin α sample contained two members of thepurothionin family when examined with SDS-polyacrylamide gelelectrophoresis. The purothionin α-1 and β samples were both homogeneousin SDS-polyacrylamide gel electrophoresis.

Routine Method Steps

Enzyme Activation Assays

The NADP-MDH, FBPase, NTR and Thioredoxin h assay methods were accordingto Florencio, F. J. et al. (1988), Arch. Biochem. Biophys. 266:496–507with slight modifications as indicated. For enzyme activation assays,the preincubation time was 20 min unless specified otherwise.

mBBr Fluorescent Labeling and SDS-polyacrylamide Gel ElectrophoresisAnalyses

Direct reduction of the proteins under study was determined by amodification of the method of Crawford et al. (Crawford, N. A. et al.(1989), Arch. Biochem. Biophys. 271:223–239). The reaction was carriedout in 100 mM potassium phosphate buffer, pH 7.1, containing 10 mM EDTAand 16% glycerol in a final volume of 0.1 ml. As indicated, 0.7 μg (0.1μM) NTR and 1 μg. (0.8 μM) thioredoxin (both routinely from E. coli wereadded to 70 μl of the buffer solution containing 1 mM NADPH and 10 μg (2to 17 μM) of target protein. When thioredoxin was reduced bydithiothreitol (DTT, 0.5 mM), NADPH and NTR were omitted. Assays withreduced glutathione were performed similarly, but at a finalconcentration of 1 mM. After incubation for 20 min, 100 nmoles of mBBrwere added and the reaction was continued for another 15 min. To stopthe reaction and derivatize excess mBBr, 10 μl of 10% SDS and 10 μl of100 mM β-mercaptoethanol were added and the samples were then applied tothe gels. In the case of reduction by glutaredoxin, the thioredoxin andNTR were-replaced by 1 μg (0.8 μM) E. coli glutaredoxin, 1.4 μg (0.14μM) glutathione reductase purified from spinach leaves (Florencio, F. J.et al. (1988), Arch. Biochem. Biophys. 266:496–507) and 1.5 mM NADPH wasused.

Gels (17.5% w/v, 1.5 mm thickness) were prepared according to Laerumnli(Laemmli, U. K. (1970), Nature 227:680–685) and developed for 16 hr. atconstant current (9 mA). Following electrophoresis, gels were placed ina solution of 40% methanol and 10% acetic acid, and soaked for 4 to 6hours with several changes of the solution. Gels were then examined forfluorescent bands with near ultraviolet light and photographed (exposuretime 25 sec) according to Crawford et al. (Crawford, N. A. et al.(1989), Arch. Biochem. Biophys. 271:223–239). Finally, gels were stainedwith Coomassie Blue and destained as before (Crawford, N. A. et al.(1989), Arch. Biochem. Biophys. 271:223–239).

Quantification of Labeled Proteins

To obtain a quantitative indication of the extent of reduction of testproteins by the NADP/thioredoxin system, the intensities of theirfluorescent bands seen in SDS-polyacrylamide gel electrophoresis wereevaluated, using a modification of the procedure of Crawford et al.(Crawford, N. A. et al. (1989), Arch. Biochem. Biophys. 271:223–239).The photographic negatives were scanned using a Pharmacia Ultrascanlaser densitometer, and the area underneath the peaks was quantitated bycomparison to a standard curve determined for each protein. For thelatter determination, each protein (at concentrations ranging from 1 to5 μg) was reduced by heating for 3 min at 100° C. in the presence of 0.5mM DTT. Labeling with mBBr was then carried out as described aboveexcept that the standards were heated for 2 min at 100° C. after thereaction was stopped with SDS and excess mBBr derivatized withβ-mercaptoethanol. Because the intensity of the fluorescent bands wasproportional to the amounts of added protein, it was assumed thatreduction was complete under the conditions used.

EXAMPLE 1 Thiorecioxin-linked Reduction of α-Amylase Inhibitors

Enzyme Activation Assays

The capability to replace a specific thioredoxin in the activation ofchloroplast enzymes is one test for the ability of thiol groups of agiven protein to undergo reversible redox change. Even though notphysiological in the case of extraplastidic proteins, this test hasproved useful in several studies. A case in point is purothionin which,when reduced by thioredoxin h activates chloroplast FBPase (Wada, K. etal. (1981), FEBS Lett. 124:237–240, and Johnson, T. C. et al. (1987),Plant Physiol. 85:446–451). The FBPase, whose physiological activator isthioredoxin f, is unaffected by thioredoxin h. In this Example, theability of cystine-rich proteins to activate FBPase as well as NADP-MDHwas tested as set forth above. The α-amylase inhibitors from durum wheat(DSG-1 and DSG-2) were found to be effective in enzyme activation;however, they differed from purothionin in showing a specificity forNADP-MDH rather than FBPase (Table I). The α-amylase inhibitors wereactive only in the presence of reduced thioredoxin h, which itself didnot significantly activate NADP-MDH under these conditions. DSG-1 andDSG-2 activated NADP-malate dehydrogenase in the presence of DTT-reducedthioredoxin h according to the reaction sequence(DTT→Thioredoxin→DSG→NADP-MDH).

The complete system for activation contained in 200 μl of 100 mMTris-HCl buffer, pH 7.9 was 10 mM DTT, 0.7 μg corn leaf NADP-MDH, 0.25μg wheat thioredoxin h and 10 μg of DSG-1 or DSG-2. In one study 20 mMβ-mercaptoethanol (β-MET) was used instead of DTT. Followingpreincubation, NADP-MDH was assayed spectrophoto-metrically.

In the enzyme activation assays, thioredoxin h was reduced by DTT; asexpected, monothiols such as β-mercaptoethanol (β-MET), which do notreduce thioredoxin at a significant rate under these conditions(Jacquot, J.-P. et al. (1981), Plant Physiol. 68:300–304; Nishizawa, A.N. et al. (1982), “Methods in Chloroplast Molecular Biology”, (M.Edelman, R. B. Hallick and N.-H. Chua, eds.) pp. 707–714, ElsevierBiomedical Press, New York, and Crawford, N. A. et al. (1989), Arch.Biochem. Biophys. 271:223–239), did not replace DTT.

NADP-MDH activity was proportional to the level of added DSG-1 and DSG-2at a constant thioredoxin h concentration. The same DTT formula was usedas set forth above. Except for varying the DSG-1 or DSG-2concentrations, conditions were identical to those previously described.When tested at a fixed DSG concentration, NADP-MDH showed enhancedactivity with increasing thioredoxin h. Except for varying thethioredoxin h concentration, conditions were as described above.

CM-1—the bread wheat protein that is similar to DSG proteins but has alower molecular weight—also activated NADP-MDH and not FBPase when 20 μgof CM-1 were used as shown in Table I. The results indicate thatthioredoxin h reduces a variety of α-amylase inhibitors, which, in turn,activate NADP-MDH in accordance with equations 4–6. These proteins wereineffective in enzyme activation when DTT was added in the absence ofthioredoxin.

$\begin{matrix} {{DTT}_{red} + {{Thioredoxin}\mspace{20mu}{\underset{\_}{h}}_{ox}}}arrow{{{Thioredoxin}\mspace{20mu}{\underset{\_}{h}}_{red}} + {DTT}_{ox}}  & (4) \\{ {{\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{ox}} + {{Thioredoxin}\mspace{20mu}{\underset{\_}{h}}_{red}}}arrow\mspace{256mu}{{\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{red}} + {{Thioredoxin}\mspace{20mu}{\underset{\_}{h}}_{ox}}} \mspace{284mu}} & (5) \\{ {{\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{red}} + {{NADP}\text{-}{MDH}_{ox}}}arrow\mspace{275mu}{{({Inactive})\mspace{11mu}\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{ox}} + {{NADP}\text{-}{{MDH}_{red}({Active})}}} \mspace{121mu}} & (6)\end{matrix}$

TABLE I

Effectiveness of Thioredoxin-Reduced Trypsin Inhibitors, Thionins, andα-Amylase Inhibitors in Activating Chloroplast NADP-Malate Dehydrogenaseand Fructose Bisphosphatase (DTT→Thioredoxin→Indicated Protein→TargetEnzyme)

Activation of NADPH-MDH was carried out as described above in thisExample except that the quantity of DSG or the other proteins tested was20 μg. FBPase activation was tested using the standard DTT assay with 1μg of E. coli thioredoxin and 20 μg of the indicated proteins. The abovevalues are corrected for the limited activation seen with E. colithioredoxin under these conditions.

No. of *ACTIVITY, nkat/mg Protein M_(r),kDa S-S Groups NADP-MDH FBPaseα-Amylase Inhibitors **DSG-2 17 5 2 0 **DSG-1 14 5 2 0 ‡CM-1 12 5 12 0Trypsin Inhibitors Cystine-rich (plant) Corn kernel 12 5 5 0 SoybeanBowman-Birk 8 7 3 0 Other types Ovomucoid 28 9 2 0 Soybean Kunitz 20 2 20 Ovoinhibitor 49 14 1 0 Bovine lung (Aprotinin) 7 3 Trace 2 Thionins**Purothionin-α₁ 6 4 1 39 **Purothionin-β 6 4 Trace 5 ‡Purothionin-α 6 40 14 *These values compare to the corresponding values of 40 and 550obtained, respectively, with spinach chloroplast thioredoxin m(NADP-MDH) and thioredoxin f. **From Durum wheat ‡From bread wheat

EXAMPLE 2 DTNB Reduction Assays

A second test for thiol redox activity is the ability to catalyze thereduction of the sulfhydryl reagent, 2′, 5′-dithiobis(2-nitrobenzoicacid) (DTNB), measured by an increase in absorbance at 412 nm. Here, theprotein assayed was reduced with NADPH via NTR and a thioredoxin. TheDTNB assay proved to be effective for the α-amylase inhibitors from bothdurum (DSG-1 and 2) and bread wheat (CM-1). When reduced by theNADP/thioredoxin system (in this case using NTR and thioredoxin from E.coli), either DSG-1 or DSG-2 markedly enhanced the reduction of DTNB(NADPH→NTR→Thioredoxin→DSG→DTNB). The DTNB reduction assay was carriedout with 10 μg thioredoxin and 10 μg NTR and 20 μg of DSG-1 or DSG-2.CM-1 was also effective in the DTNB reduction assay, and, as withNADP-MDH activation (Table I), was detectably more active than the DSGproteins The conditions for the CM-1 assay were the same as for theDSG/DTNB assay except that the DSG proteins were omitted and purothioninα, 20 μg or CM-1, 20 μg was used). The results thus confirmed the enzymeactivation experiments in Example 1 and showed that the α-amylaseinhibitors can be reduced physiologically by the NADP/thioredoxinsystem. The role of the α-amylase inhibitors in promoting the reductionof DTNB under these conditions is summarized in equations 7–9.

$\begin{matrix}{{NADPH} + {{Thioredoxin}_{ox}\overset{NTR}{\longrightarrow}{Thioredoxin}_{red}} + {NADP}} & (7) \\ {{Thioredoxin}_{red} + {\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{ox}}}arrow\mspace{115mu}{{Thioredoxin}_{ox} + {\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{{red}\mspace{205mu}}}}  & (8) \\{ {{\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{red}} + {DTNB}_{ox}}arrow{{\alpha\text{-}{Amylase}\mspace{14mu}{inhibitor}_{ox}} + {DTNB}_{red}} \mspace{214mu}} & (9)\end{matrix}$

EXAMPLE 3 Protein Reduction Measurements

The availability of monobromobimane (mBBr) and its adaptation for use inplant systems has given a new technique for measuring the sulfhydrylgroups of plant proteins (Crawford, N. A. et al. (1989), Arch. Biochem.Biophys. 271:223–239). When coupled with SDS-polyacrylamide gelelectrophoresis, mBBr can be used to quantitate the change in thesulfhydryl status of redox active proteins, even in complex mixtures.This technique was therefore applied to the inhibitor proteins toconfirm their capacity for reduction by thioredoxin. Here, the testprotein was reduced with thioredoxin which itself had been previouslyreduced with either DTT or NADPH and NTR. The mBBr derivative of thereduced protein was then prepared, separated from other components bySDS-polyacrylamide gel electrophoresis and its reduction state wasexamined by fluorescence. In the experiments described below,thioredoxin from E. coli was found to be effective in the reduction ofeach of the targeted proteins. Parallel experiments revealed thatthioredoxin h and calf thymus thioredoxins reduced, respectively, theproteins from seed and animal sources.

In confirmation of the enzyme activation and dye reduction experiments,DSG-1 was effectively reduced in the presence of thioredoxin. Followingincubation the proteins were derivatized with mBBr and fluorescencevisualized after SDS-polyacrylamide gel electrophoresis. Reduction wasmuch less with DTT alone and was insignificant with GSH. A similarrequirement for thioredoxin was observed for the reduction of CM-1 andDSG-2 (data not shown). While the thioredoxin used was from E. colisimilar results were obtained with wheat thioredoxin h. Thioredoxin wasalso required when DTT was replaced by NADPH and NTR (data not shown).

EXAMPLE 4 Thioredoxin-linked Reduction of Cystine-Rich Plant TrypsinInhibitors

Whereas the major soluble cystine-rich proteins of wheat seeds can actas inhibitors of exogenous α-amylases, the cystine-rich proteins of mostother seeds lack this activity, and, in certain cases, act as specificinhibitors of trypsin from animal sources. While these proteins can bereduced with strong chemical reductants such as sodium borohydride(Birk, Y. (1985), Int. J. Peptide Protein Res. 25:113–131, and Birl, Y.(1976), Meth. Enzymol. 45:695–7390), there is little evidence that theycan be reduced under physiological conditions. It was therefore ofinterest to test trypsin inhibitors for the capacity to be reduced bythioredoxin. The cystine-rich representatives tested included thesoybean Bowman-Birk and corn kernel trypsin inhibitors. The results inboth cases were positive: each inhibitor activated NADP-MDH (but notFBPase) when added in the presence of DTT-reduced thioredoxin (Table I)and each reduced DTNB in the presence of NADPH, NTR and thioredoxin(data not shown).

As found for the α-amylase inhibitors, the thioredoxin-dependentreduction of the cystine-rich trypsin inhibitors could be directlymonitored by the mBBr/SDS-polyacrylamide gel electrophoresis technique.Thus, significant reduction by DTT was observed only in the presence ofreduced thioredoxin with both the Bowman-Birk (BBTI) inhibitor whichshowed a highly fluorescent fast moving band and corn kernel (CKTI)trypsin inhibitor which showed a highly fluorescent band migratingbehind thioredoxin.

EXAMPLE 5 Thioredoxin-linked Reduction of Other Trypsin Inhibitors andPurothionins

In view of the finding that cystine-rich trypsin inhibitors from seedscan undergo specific reduction by thioredoxin, the question arose as towhether other types of trypsin inhibitor proteins share this property.In the course of this study, several such inhibitors—soybean Kunitz,bovine lung aprotinin, egg white ovoinhibitor and ovomucoid trypsininhibitors—were tested. While the parameters tested were not asextensive as with the cystine-rich proteins described above, it wasfound that the other trypsin inhibitors also showed a capacity to bereduced specifically by thioredoxin as measured by both the enzymeactivation and mBBr/SDS-polyacrylamide gel electrophoresis methods. Aswas the case for the cystine-rich proteins described above, the trypsininhibitors tested in this phase of the study (soybean Kunitz and animaltrypsin inhibitors) activated NADP-MDH but not FBPase (Table I). Bovinelung aprotinin was an exception in that it activated FBPase moreeffectively than NADP-MDH. It might also be noted that aprotininresembles certain of the seed proteins studied here in that it shows ahigh content of cystine (ca. 10%) (Kassel, B. et al. (1965), Biochem.Biophys. Res. Commun. 20:463–468).

The fluorescence evidence for the thioredoxin-linked reduction of one ofthese proteins, the Kunitz inhibitor, was shown by a highly fluorescentslow moving band in an mBBr/SDS-polyacrylamide electrophoretic gel. Inits reduced form, the Kunitz inhibitor also yielded a fluorescent fastmoving band. The nature of this lower molecular mass species is notknown. Its position on the gel suggests that it could representBowman-Birk inhibitor present as a contaminant in the Kunitzpreparation; however, such a component was not evident in Coomassie Bluestained SDS gels. The animal inhibitors which yielded a singlefluorescent band of the expected molecular weight, also showed athioredoxin requirement for reduction (data not shown).

In confirmation of earlier results, thioredoxin-reduced purothioninconsistently activated FBPase and the type tested earlier,purothionin-α, failed to activate NADP-MDH (Table I) (Wada, K. et al.(1981), FEBS Lett. 124:237–240). However, in contrast to purothionin-αfrom bread wheat, two purothionins previously not examined (purothioninsα-1 and β from durum wheat) detectably activated NADP-MDH (Table I). Thetwo durum wheat Durothionins also differed in their abilitv to activateFBPase. The activity differences between these purothionins wereunexpected in view of the strong similarity in their amino acidsequences (Jones, B. L. et al. (1977), Cereal Chem. 54:511–523) and intheir ability to undergo reduction by thioredoxin. A requirement forthioredoxin was observed for the reduction of purothionin (here theα-type) by the SDS-PAGE fluorescence procedure.

EXAMPLE 6 Quantitation of Reduction

The above Examples demonstrate that thioredoxin reduces a variety ofproteins, including α-amylase, such as the CM and DSG inhibitors, andtrypsin inhibitors from seed as well as animal sources. While clear inthe qualitative sense, the above results give no quantitative indicationof the extent of reduction. Therefore, an experiment was conductedfollowing the protocol of Crawford et al. (Crawford, N. A. et al.(1989), Arch. Biochem. Biophys. 271:223–239).

As shown in Table II, the extent of reduction of the seed inhibitorproteins by the E. coli NADP/thioredoxin system was time-dependent andreached, depending on the protein, 15 to 48% reduction after two hours.The results, based on fluorescence emitted by the major proteincomponent, indicate that thioredoxin acts catalytically in the reductionof the α-amylase and trypsin inhibitors. The ratio of protein reducedafter two hours to thioredoxin added was greater than one for both themost highly reduced protein (soybean Bowman-Birk trypsin inhibitor) andthe least reduced protein (corn kernel trypsin inhibitor)—i.e.,respective ratios of 7 and 2 after a two-hour reduction period. Itshould be noted that the values in Table II were obtained under standardassay conditions and no attempt was made to optimize reduction bymodifying those conditions.

TABLE II Extent of Reduction of Seed Proteins by the NADP/ThioredoxinSystem Using the mBBr/SDS-Polyacrylamide Gel Electrophoresis Procedure %Reduction After Protein 20 min 120 min Purothionin-β 15 32 DSG-1 22 38Corn kernel trypsin inhibitor 3 15 Bowman-Birk trypsin inhibitor 25 48Kunitz trypsin inhibitor 14 22 The following concentrations of proteinswere used (nmoles): thioredoxin, 0.08; NTR, 0.01; purothionin-β, 1.7;DSG-1, 0.7; corn kernel trypsin inhibitor, 1.0; Bowman-Birk trypsininhibitor, 1.3; and Kunitz trypsin inhibitor, 0.5. Except for theindicated time difference, other conditions were as in Examples 1–4.

EXAMPLE 7 E. coli Glutaredoxin as Reductant

Bacteria and animals are known to contain a thiol redox protein,glutaredoxin, that can replace thioredoxin in reactions such asribonucleotide reduction (Holmgren, A. (1985), Annu. Rev. Biochem.54:237–271). Glutaredoxin is reduced as shown in equations 10 and 11.

$\begin{matrix}{{{NADPH} + {GSSG}}\mspace{14mu}\underset{\text{reductase}}{\overset{\text{~~~Glutathione~~}\;}{arrow}}\mspace{14mu}{{2\mspace{20mu}{GSH}} + {NADP}}} & (10)\end{matrix}$2 GSH+Glutaredoxino_(ox)→GSSG+Glutaredoxin_(red)  (11)

So far there is no evidence that glutaredoxin interacts with proteinsfrom higher plants. This ability was tested, using glutaredoxin from E.coli and the seed proteins currently under study. Reduction activity wasmonitored by the mBBr/SDS polyacrylamide gel electrophoresis procedurecoupled with densitometric scanning. It was observed that, under theconditions previously described, glutaredoxin could effectively replacethioredoxin in some, but not all cases. Thus, glutaredoxin was found tobe active in the reduction of the following (the numbers indicate thepercentage reduction relative to E. coli thioredoxin): DSG-1 and CM-1α-amylase inhibitors (147 and 210%, respectively); corn kernel trypsininhibitor (424%); and purothionin (82, 133, and 120% for the α, α1 and βforms, respectively). Glutaredoxin was ineffective in the reduction ofthe DSG-2 α-amylase inhibitor and the soybean Bowman-Birk and Kunitztrypsin inhibitors. The trypsin inhibitors from animal sources alsoshowed a mixed response to glutaredoxin. Egg white ovoinhibitor waseffectively reduced (55% reduction relative to E. coli thioredoxin)whereas egg white ovomucoid inhibitor and bovine lung aprotinin were notaffected. Significantly, as previously reported (Wolosiuk, R. A. et al.(1977), Nature 266:565–567), glutaredoxin failed to replace thioredoxinas the immediate reductant in the activation of thioredoxin-linkedenzymes of chlioroplasts, FBPase and NADP-MDH (data not shown).

The above Examples demonstrate that some of the enzyme inhibitorproteins tested can be reduced by glutaredoxin as well as thioredoxin.Those specific for thioredoxin include an α-amylase inhibitor (DSG-2),and several trypsin inhibitors (Kunitz, Bowman-Birk, aprotinin, andovomucoid inhibitor). Those proteins that were reduced by eitherthioredoxin or glutaredoxin include the purothionins, two α-amylaseinhibitors (DSG-1, CM-1), a cystine-rich trypsin inhibitor from plants(corn kernel inhibitor), and a trypsin inhibitor from animals (egg whiteovoinhibitor) These results raise the question of whether glutaredoxinoccurs in plants. Glutaredoxin was reported to be present in a greenalga (Tsang, M. L.-S. (1981), Plant Physiol. 68:1098–1104) but not inhigher plants.

Although the activities of the NADP-MDH and FBPase target enzymes shownin Table I are low relative to those seen following activation by thephysiological chloroplast proteins (thioredoxin m or f), the valuesshown were found repeatedly and therefore are considered to be real. Itseems possible that the enzyme specificity shown by the inhibitorproteins, although not relevant physiologically, reflects a particularstructure achieved on reduction. It remains to be seen whether such areduced structure is related to function within the seed or animal cell.

The physiological consequence of the thioredoxin (or glutaredoxin)linked reduction event is of considerable interest as the function ofthe targeted proteins is unclear. The present results offer a newpossibility. The finding that thioredoxin reduces a wide variety ofinhibitor proteins under physiological conditions suggests that, in theabsence of compartmental barriers, reduction can take place within thecell.

EXAMPLE 8 Inactivation of Soybean Trypsin Inhibitor in Soybean Meal

The goal of this Example is to inactivate the Bowman-Birk and Kunitztrypsin inhibitors of soybeans, The following protocol applies to animalfeed preparations. To 10 g of soybean meal are added 0.2 μg thioredoxin,0.1 μg NADP-thioredoxin reductase and 500 nanamoles NADPH together with1 M Tris-HCl buffer, pH 7.9, to give 5.25 ml of 30 mM Tris-HCl. Theabove mixture is allowed to sit for about 30 min at room temperature.Direct reduction of the soybean trypsin inhibitor is determined usingthe mBBr fluorescent labeling/SDS-polyacrylamide gel electrophoresismethod previously described (Kobrehel, K. et al. (1991), J. Biol. Chem.266:16135–16140). An analysis of the ability of the treated flour fortrypsin activity is made using modifications of the insulin and BAEE(Na-benzoyl-L-arginine ethyl ester) assays (Schoellmann, G. et al.(1963), Biochemistry 252:1963; Gonias, S. L. et al. (1983), J. Biol.Chem. 258:14682). From this analysis it is determined that soybean mealso treated with the NADP/thioredoxin system does not inhibit trypsin.

EXAMPLE 9 Inactivation of α-Amylase Inhibitors in Cereals

To 10 g of barley malt are added 0.2 μg thioredoxin, 0.1 μgNADP-thioredoxin reductase and 500 nanamoles NADPH together with 1 MTris-HCl buffer, pH 7.9, to give 5.25 ml of 30 mM Tris-HCl. The abovemixture is allowed to sit for about 30 min at room temperature. Directreduction of the α-amylase inhibitors is determined using the mBBrfluorescent labeling/SDS-polyacrylamide gel electrophoresis methodpreviously described (Kobrehel, K. et al. (1991), J. Biol. Chem.266:16135–16140). α-Amylase activity is monitored by following therelease of maltose from starch (Bernfeld, P. (1955), Methods in Enzymol.1:149). From this analysis it is determined that barley so treated withthe NADP/thioredoxin system does not inhibit α-amylase.

REDUCTION OF CEREAL PROTEINS Materials and Methods

Plant Material

Seeds and semolina of durum wheat (Triticum durum, Desf. cv. Monroe)were kind gifts of Dr. K. Kahn.

Germination of Wheat Seeds

Twenty to 30 seeds were placed in a plastic Petri dish on three layersof Whatman #1 filter paper moistened with 5 ml of deionized water.Germination was carried out for up to 4 days at room temperature in adark chamber.

Reagents/Fine Chemicals

Biochemicals and lyophilized coupling enzymes were obtained from SigmaChemical Co. (St. Louis, Mo.). E. coli thioredoxin and NTR werepurchased from American Diagnostica, Inc. (Greenwich, Conn.). Wheatthioredoxin h and NTR were isolated from germ, following the proceduresdeveloped for spinach leaves (Florencio, F. J. et al. (1988), Arch.Biochem. Biophys. 266:496–507). E. coli glutaredoxin was a kind gift ofProfessor A. Holmgren. Reagents for SDS-polyacrylamide gelelectrophoresis were purchased from Bio-Rad Laboratories (Richmond,Calif.). Monobromobimane (mBBr) or Thiolite was obtained from CalbiochemCo. (San Diego, Calif.). Aluminum lactate and methyl green were productsof Fluka Chemicals Co. (Buchs, Switzerland).

Gliadins and Glutenins

For isolation of insoluble storage proteins, semolina (0.2 g) wasextracted sequentially with 1 ml of the following solutions for theindicated times at 25° C.: (1) 50 mM Tris-HCl, pH 7.5 (20 min); (2) 70%ethanol (2 hr); and (3) 0.1 M acetic acid (2 hr). During extraction,samples were placed on an electrical rotator and, in addition,occasionally agitated with a vortex mixer. After extraction with eachsolvent, samples were centrifuged (12,000 rpm for 5 min) in an Eppendorfmicrofuge and, supernatant fractions were saved for analysis. In betweeneach extraction, pellets were washed with 1 ml of water, collected bycentrifugation as before and the supernatant wash fractions werediscarded. By convention, the fractions are designated: (1)albumin/globulin; (2) gliadin; and (3) glutenin.

In vitro mBBr Labelling of Proteins

Reactions were carried out in 100 mM Tris-HCl buffer, pH 7.9. Asindicated, 0.7 μg NTR and 1 μg thioredoxin (both from E. coli unlessspecified otherwise) were added to 70 μl of this buffer containing 1 mMNADPH and 10 μg of target protein. When thioredoxin was reduced bydithiothreitol (DTT), NADPH and NTR were omitted and DTT was added to0.5 mM. Assays with reduced glutathione were performed similarly, but ata final concentration of 1 mM. After incubation for 20 min, 100 nmolesof mBBr were added and the reaction was continued for another 15 min. Tostop the reaction and derivatize excess mBBr, 10 μl of 10% SDS and 10 μlof 100 mM β-mercaptoethanol were added and the samples were then appliedto the gels. For reduction by glutaredoxin, the thioredoxin and NTR werereplaced by 1 μg E. coli glutaredoxin, 1.4 μg glutathione reductase(purified from spinach leaves) and 1.5 mM NADPH.

In vivo mBBr Labelling of Proteins

At the indicated times, the dry seeds or germinating seedlings (selectedon the basis of similar radical length) were removed from the Petri dishand their embryos or germinated axes were removed. Five endosperms fromeach lot were weighed and then ground in liquid N₂ with a mortal andpestle. One ml of 2.0 mM mBBr in 100 mM Tris-HCl, pH 7.9, buffer wasadded just as the last trace of liquid N₂ disappeared. The thawedmixture was then ground for another minute and transferred to amicrofuge tube. The volume of the suspension was adjusted to 1 ml withthe appropriate mBBr or buffer solution. Protein fractions ofalbumin/globulin, gliadin and glutenin were extracted from endosperm ofgerminated seedlings as described above. The extracted protein fractionswere stored at −20° C. until use. A buffer control was included for eachtime point.

SDS-Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide electrophoresis of the mBBr-derivatized samples wasperformed in 15% gels at pH 8.5 as described by Laemmli, U. K. (1970),Nature 227:680–685. Gels of 1.5 mm thickness were developed for 16 hr.at a constant current of 9 mA.

Native Gel Electrophoresis

To resolve the different types of gliadins, native polyacrylamide gelelectrophoresis was performed in 6% gels (a procedure designed toseparate gliadins into the four types) as described by Bushuk andZillman (Bushuk, W. et al. (1978), Can. J. Plant Sci. 58:505–515) andmodified for vertical slab gels by Sapirstein and Bushuk (Sapirstein, H.D. et al. (1985), Cereal Chem. 62:372–377). A gel solution in 100 mlfinal volume contained 6.0 g acrylamide, 0.3 g bisacrylamide, 0.024 gascorbic acid, 0.2 mg ferrous sulfate heptahydrate and 0.25 g aluminumlactate. The pH was adjusted to 3.1 with lactic acid. The gel solutionwas degassed for 2 hr. on ice and then 0.5 ml of 3% hydrogen peroxidewas added as a polymerization catalyst. The running buffer, alsoadjusted to pH 3.1 with lactic acid, contained 0.5 g aluminum lactateper liter. Duration of electrophoresis was approximately 4 hr., with aconstant current of 50 mA. Electrophoresis was terminated when thesolvent front, marked with methyl green tracking dye, migrated to about1 cm from the end of the gel.

mBBr Removal/Fluorescence Photography

Following electrophoresis, gels were placed in 12% (w/v) trichloroaceticacid and soaked for 4 to 6 hr. with one change of solution to fix theproteins; gels were then transferred to a solution of 40% methanol/10%acetic acid for 8 to 10 hr. to remove excess mBBr. The fluorescence ofmBBr, both free and protein bound, was visualized by placing gels on alight box fitted with an ultraviolet light source (365 nm). Followingremoval of the excess (free) mBBr, gels were photographed with PolaroidPositive/Negative Landfilm, type 55, through a yellow Wratten gelatinfilter No. 8 (cutoff=460 nm) (exposure time ranged from 25 to 60 sec atf4.5) (Crawford, N. A. et al. (1989), Arch. Biochem. Biophys.271:223–239).

Protein Staining/Destaining/Photography

SDS-gels were stained with Coomassie Brilliant Blue R-250 in 40%methanol/10% acetic acid for 1 to 2 hr. and destained overnight asdescribed before (Crawford, N. A. et al. (1989), Arch. Biochem. Biophys.271:223–239). Aluminum lactate native gels were stained overnight in afiltered solution containing 0.1 g Coomassie Brilliant Blue R-250(dissolved in 10 ml 95% ethanol) in 240 ml 12% trichloroacetic acid.Gels were destained overnight in 12% trichloroacetic acid (Bushuk, W. etal. (1978), Can. J. Plant Sci. 58:505–515, and Sapirstein, H. D. et al.(1985), Cereal Chem. 62:372–377).

Protein stained gels were photographed with Polaroid type 55 film toproduce prints and negatives. Prints were used to determine bandmigration distances and loading efficiency.

The Polaroid negatives of fluorescent gels and prints of wet proteinstained gels were scanned with a laser densitometer (Pharmacia-LKBUltroScan XL). Fluorescence was quantified by evaluating peak areasafter integration with GelScan XL software.

Enzyme Assays

The following activities were determined in crude extracts withpreviously described methods: hexokinase (Baldus, B. et al. (1981),Phytochem. 20:1811–1814), glucose-6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase (Schnarrenberger, C. et al. (1973),Arch. Biochem. Biophys. 154:438–448), glutathione reductase, NTR andthioredoxin h (Florencio, F. J. et al. (1988), Arch. Biochiem. Biophys.266:496–507).

Protein Determination

Protein concentrations were determined by the Bradford method (Bradford,M. (1976) Anal. Biochem. 72:248–256), with Bio-Rad reagent and bovineserum albumin as a standard.

Subunit Molecular Weight Determination

The subunit molecular weight of gliadins and glutenins was estimated onSDS-PAGE gels by using two sets of molecular weight standards (kDa). Thefirst set consisted of BSA (66), ovalbumin (45), soybean trypsininhibitor (20.1), myoglobin (17), cytochrome c (12.4) and aprotinin(6.5). The other set was the BioRad Prestained Low SDS-PAGE standards:phosphorylase b (110), BSA (84), ovalbumin (47), carbonic anhydrase(33), soybean trypsin inhibitor (24) and lysozyme (16).

EXAMPLE 10 Reduction of Gliadins

As a result of the pioneering contributions of Osborne and coworkers acentury ago, seed proteins can be fractionated on the basis of theirsolubility in aqueous and organic solvents (20). In the case of wheat,preparations of endosperm (flour or semolina) are historicallysequentially extracted with four solutions to yield the indicatedprotein fraction: (i) water, albumins; (ii) salt water, globulins; (iii)ethanol/water, gliadins; and (iv) acetic acid/water, glutenins. A widebody of evidence has shown that different proteins are enriched in eachfraction. For example, the albumin and globulin fractions containnumerous enzymes, and the gliadin and glutenin fractions are in thestorage proteins required for germination.

Examples 1, 4 and 5 above describe a number of water soluble seedproteins (albumins/globulins, e.g., α-amylase inhibitors, cystine-richtrypsin inhibitors, other trypsin inhibitors and thionines) that arereduced by the NADP/thioredoxin system, derived either from the seeditself or E. coli. The ability of the system to reduce insoluble storageproteins from wheat seeds, viz., representatives of the gliadin andglutenin fractions, is described below. Following incubation with theindicated additions, the gliadin proteins were derivatized with mBBr andfluorescence was visualized after SDS-polyacrylamide gelelectrophoresis. The lanes in this first gliadin gel were as follows: 1.Control: no addition. 2. GSH/GR/NADPH: reduced glutathione, glutathionereductase (from spinach leaves) and NADPH. 3. NGS: NADPH, reducedglutathione, glutathione reductase (from spinach leaves) andglutaredoxin (from E. coli). 4. NTS: NADPH, NTR, and thioredoxin (bothproteins from E. coli). 5. MET/T(Ec): β-mercaptoethanol and thioredoxin(E. coli). 6. DTT. 7. DTT/T(Ec): DTT and thioredoxin (E. coli). 8.DTT/T(W): Same as 7 except with wheat thioredoxin h. 9. NGS,-Gliadin:same as 3 except without the gliadin protein fraction. 10. NTS,-Gliadin:same as 4 except without the gliadin protein fraction. Based on itsreactivity with mBBr, the gliadin fraction was extensively reduced bythioredoxin. The major members undergoing reduction showed a Mr rangingfrom 25 to 45 kDa. As seen in Examples 1, 4 and 5 with the seedα-amylase and trypsin inhibitor proteins, the gliadins were reduced byeither native h or E. coli type thioredoxin (both homogeneous); NADPH(and NTR) or DTT could serve as the reductant for thioredoxin. Much lessextensive reduction was observed with glutathione and glutaredoxin—aprotein able to replace thioredoxin in certain E. coli and mammalianenzyme systems, but not known to occur in higher plants.

The gliadin fraction is made up of four different protein types,designated α, β, γ and ω, which can be separated by nativepolyacrylamide gel electrophoresis under acidic conditions (Bushuk, W.et al. (1978), Can. J. Plant Sci. 58:505–515; Kasarda, D. D. et al.(1976), Adv. Cer. Sci. Tech. 1:158–236; Sapirstein, H. D. et al. (1985),Cereal Chem. 62:372–377; and Tatham, A. S. et al. (1990), Adv. Cer. Sci.Tech. 10:1–78). Except for the ω gliadins, each species contains cystine(S—S) groups and thus has the potential for reduction by thioredoxin. Inthis study, following incubation with the indicated additions, proteinswere derivatized with mBBr, and fluorescence was visualized afteracidic-polyacrylamide gel electrophoresis. The lanes in the secondgliadin gel in this study were as follows: 1. Control: no addition. 2.GSH: reduced glutathione. 3. GSH/GR/NADPH: reduced glutathione,glutathione reductase (from spinach leaves) and NADPH. 4. NGS: NADPH,reduced glutathione, glutathione reductase (from spinach leaves) andglutaredoxin (from E. coli). 5. NGS+NTS: combination of 4 and 6. 6. NTS:NADPH, NTR, and thioredoxin (both proteins from E. coli). 7. MET/T(Ec):β-mercaptoethanol and thioredoxin (E. coli). 8. DTT/T(Ec): DTT andthioredoxin (E. coli). 9. NTS(-T): same as 6 except without thioredoxin.10. NGS+NTS,-Gliadin: same as 5 except without the gliadin fraction.

When the thioredoxin-reduced gliadin fraction was subjected to nativegel electrophoresis, the proteins found to be most specifically reducedby thioredoxin were recovered in the α fraction. There was activereduction of the β and γ gliadins, but as evident from the densitometerresults summarized in Table III, the reduction within these groups wasnonspecific, i.e., relatively high levels of reduction were alsoachieved with glutathione and glutaredoxin. There was especially strongreduction of the γ gliadins by DTT-reduced thioredoxin. As anticipated,there was no reduction of the ω gliadins. The evidence indicates thatthioredoxin (either native h or E. coli) specifically reduces certain ofthe gliadins, especially the α type.

TABLE III Reductant Specificity of the Different Types of GliadinsGliadin, % Relative Reduction Reductant α β γ Aggregate* None 22.4 30.424.3 29.2 Glutathione 36.4 68.1 60.6 60.1 Glutaredoxin 43.5 83.3 79.761.5 Thioredoxin 100.0 100.0 100.0 100.0 *Proteins not entering the gelThe area under α, β, γ and aggregate peaks following reduction by theNADP/thioredoxin system were: 4.33, 8.60, 5.67 and 0.74 Absorbance unitstimes millimeters, respectively. These combined areas were about 65% ofthose observed when thioredoxin was reduced by DTT with the second gel,with the reaction conditions as in Example 10.

EXAMPLE 11 Reduction of Glutenins

The remaining group of seed proteins to be tested for a response tothioredoxin—the glutenins—while the least water soluble, are perhaps ofgreatest interest. The glutenins have attracted attention over the yearsbecause of their importance for the cooking quality of flour andsemolina (MacRitchie, F. et al. (1990), Adv. Cer. Sci. Tech. 10:79–145).Testing the capability of thioredoxin to reduce the proteins of thisgroup was, therefore, a primary goal of the current investigation.

Several glutenins were reduced specifically by thioredoxin when themBBr/SDS-page technique was applied and the conditions were as inExample 10 with the first gel. The most extensive reduction was observedin the low molecular mass range (30–55 kDa). The reduction observed inthe higher molecular mass range was less pronounced, but stillobvious—especially in the 100 kDa region and above. Though not shownreduction may also occur in the 130 kDa range. Like the gliadins,certain of the glutenins were appreciably reduced by glutathione andglutaredoxin. However, in all cases, reduction was greater withthioredoxin and, in some cases, specific to thioredoxin (Table IV, noteproteins in the 30–40 and 60–110 kDa range). As observed with the otherwheat proteins tested, both the native h anal E. coli thioredoxins wereactive and could be reduced with either NADPH and the corresponding NTRor with DTT. Thus as found for the gliadins, certain glutenins werereduced in vitro specifically by thioredoxin, whereas others were alsoreduced, albeit less effectively, by glutathione and glutaredoxin.

TABLE IV Reductant Specificity of Glutenins Glutenin, % RelativeReduction* Reductant 60–110 kDa 40–60 kDa 30–40 kDa None 8 23 16Glutathione 31 51 29 Glutaredoxin 50 72 40 Thioredoxin* 100 100 100*Area under the three molecular weight classes (from high to low)following reduction by the NADP/thioredoxin system were: 1.5, 5.67 and5.04 Absorbance units times millimeters, respectively. Reactionconditions as in the Example 1 study of the effect of thioredoxin hconcentration on the activation of NADP-MDH by DSG-1 or -2 α-amylaseinhibitors.

EXAMPLE 12 In vivo Reduction Experiments

The above Example demonstrates that thioredoxin specifically reducescomponents of the wheat gliadin and glutenin fractions when tested invitro. The results, however, provide no indication as to whether theseproteins are reduced in vivo during germination—a question that, to ourknowledge, had not been previously addressed (Shutov, A. D. et al.(1987), Phytochem. 26:1557–1566).

To answer this question, the mBBr/SDS-PAGE technique was applied tomonitor the reduction status of proteins in the germinating seed. Weobserved that reduction of components in the Osborne fractions increasedprogressively with time and reached a peak after 2 to 3 daysgermination.

The observed increase in reduction ranged from 2-fold with the gliadins,to 3-fold with the albumin/globulins and 5-fold with the glutenins. Theresults suggest that, while representatives of the major wheat proteingroups were reduced during germination, the net redox change wasgreatest with the glutenins.

Although providing new evidence that the seed storage proteins undergoreduction during germination, the results give no indication as to howreduction is accomplished, i.e., by glutathione or thioredoxin. To gaininformation on this point, the in vivo reduction levels of the principalthioredoxin-linked gliadins (30–50 kDa) and glutenins (30–40, 40–60 kDa)was compared with the reduction determined from in vitro measurements(cf. Table IV). For this purpose, the ratio of fluorescence to Coomassiestained protein observed in vivo during germination and in vitro withthe appropriate enzyme reduction system was calculated. The results(principal thioredoxin linked gliadins were those in the Mr range from25 to 45 kDa, and glutenins were those in the Mr range from 30 to 60kDa) suggest that, while glutathione could account for a significantpart of the in vivo reduction of the gliadin fraction (up to 90%), thiswas not the case with the glutenins whose reduction seemed to requirethioredoxin. The level of reduction that could be ascribed toglutathione (or glutaredoxin) was insufficient to account for the levelsof reduced glutenin measured in the germinating seed.

EXAMPLE 13 Enzyme Measurements

The source of NADPH needed for the NTR linked reduction of thioredoxin hwas also investigated. Semolina was analyzed for enzymes that functionin the generation of NADPH in other systems, notably dehydrogenases ofthe oxidative phosphate pathway. The results summarized in Table Vconfirm earlier evidence that endosperm extracts contain the enzymesneeded to generate NADPH from glucose via this pathway: hexokinase,glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase(Tatham, A. S. el al. (1990), Adv. Cer. Sci. Tech. 10:1–78). It isnoteworthy that the glucose 6-phosphate dehydrogenase activity seen inTable V was insensitive to reduced thioredoxin (data not shown). In thisrespect the endosperm enzyme resembles its cytosolic rather than itschloroplast counterpart from leaves (Fickenscher, K. et al. (1986),Arch. Biochem. Biophys. 247:393–402; Buchanan, B. B. (1991), Arch.Biochem. Biophys. 288:1–9; Scheibe, R. et al. (1990), Arch. Biochem.Biophys. 274:290–297).

As anticipated from earlier results with flour (Johnson, T. C. et al.(1987), Planta 171:321–331; Suske, G. et al. (1979), Z. Naturforsch. C34:214–221), semolina also contained thioredoxin h and NTR (Table V).Interestingly, based on activity measurements, NTR appeared to be arate-limiting component in preparations from the cultivar examined.

TABLE V Activities of Enzymes Effecting the Reduction of Thioredoxin hin Semolina (Glucose→Glu-6-P→6-P-Gluconate→NADP→Thioredoxin h) ActivityProtein (nkat/mg protein) Hexokinase 0.28 Glucose-6-P dehydrogenase 0.456-P-Gluconate dehydrogenase 0.39 NTR 0.06 Thioredoxin h 0.35

The present results suggest that thioredoxin h functions as a signal toenhance metabolic processes associated with the germination of wheatseeds. Following its reduction by NTR and NADPH (generated via theoxidative pentose phosphate pathway), thioredoxin h appears to functionnot only in the activation of enzymes, but also in the mobilization ofstorage proteins.

EXAMPLE 14 Improvement of Dough Quality

Dough quality was improved by reducing the flour proteins using theNADP/thioredoxin system. Reduced thioredoxin specifically breakssulfur-sulfur bonds that cross-link different parts of a protein andstabilize its folded shape. When these cross-links are cut the proteincan unfold and link up with other proteins in bread, creating aninterlocking lattice that forms the elastic network of dough. The doughrises because the network traps carbon dioxide produced by yeast in thefermenting process. It is proposed that the reduced thioredoxinactivated the gliadins and glutenins in flour letting them recombine ina way that strengthened the dough. Reduced thioredoxin strengthened theprotein network formed during dough making. For these tests (using 10 gmof either intermediate quality wheat flour obtained from a local millerin Montpellier, France, or poor quality wheat also obtained from a localmiller in Montpellier, France, this poor quality wheat being mainly ofthe Apollo cultivar), 0.2 μg E. coli thioredoxin, 0.1 μg E. coliNADP-thioredoxin reductase and 500 nanomoles NADPH were added togetherwith 1 M Tris-HCl, pH 7.9 buffer to give 5.25 ml of a 30 mM Tris-HClenzyme system mixture. The reaction was carried out by mixing the enzymesystem mixture with the 10 gm of the flour in a micro-farinograph at 30°C. The resulting farinograph measurements showed a strengthening of thedough by the added NADP/thioredoxin system. With a flour of poorquality, the farinograph reading was stable for at least 4 min after thedough was formed in the presence of the reduction system, whereas thereading dropped immediately after dough formation in the control withoutthis addition. The improving effect was persistent and was maintainedthroughout the run. Expressed another way, the micro-farinograph readingis 375 Brabender units, 7 min after dough formation with the poorquality wheat control (no added enzyme system) versus 450 Brabenderunits for the same poor quality wheat treated with components of theNADP/thioredoxin system (NADPH, thioredoxin and NADP-thioredoxinreductase).

Another farinograph study was carried out as above with 10 gm of Apolloflour only the concentration of NADPH was 500 μmoles instead ofnanomoles. The farinograph measurements showed that this amount of NADPHalso resulted in a definite improvement in the quality of the dough.

Higher farinograph measurements of dough correspond to improved doughstrength and improved baked good characteristics such as better crumbquality, improved texture and higher loaf volume. Also, based on in vivoanalyses with the isolated proteins, the native wheat seedNADP/thioredoxin system will also be effective in strengthening thedough.

For purposes of baking and other aspects of this invention, ranges ofabout 0.1 to 3.0 μg of a thioredoxin (preferably E. coli or thioredoxinh) and from about 0.1 to 2.0 μg reductase and about 30 to 500 nanomolesof NADPH are added for about every 10 gm of flour. The optimal levels ofthioredoxin and reductase depend on flour quality. In general, thehigher the flour quality, the higher the level of thioredoxin andreductase required. Thioredoxin can also be reduced by lipoic acidinstead of by the NADPH/NADP-thioredoxin reductase reduction system. Theother dough ingredients such as milk or water are then added. However,the liquid may first be added to the NTR/thioredoxin system and thenadded to the flour.

It is preferred that yeast for purposes of leavening be added after thereduced thioredoxin has had a chance to reduce the storage proteins. Thedough is then treated as a regular dough proofed, shaped, etc. andbaked.

NADPH can be replaced in this Example as well as in the followingExamples with an NADPH generator such as one consisting of 100 μMglucose 6-phosphate, 100 μM NADP and 0.05 units (0.2 μgram) glucose6-phosphate dehydrogenase from a source such as yeast. The NADPHgenerator is added together with thioredoxin and NADP-thioredoxinreductase at the start of the dough making process.

A higher farinograph measurement was obtained when 10 gm of Apollocultivar (CV) wheat were reacted with 20 μl NADP (25 mM), 20 μl G6P (25mM), 0.25 μg G6PDase, 0.1 μg NTR and 0.2 μg thioredoxin h contained in4.25 ml H₂O and 0.90 ml Tris-HCl (30 mM, pH 7.9). A higher farinographmeasurement was also obtained when 10 gm of Apollo wheat were reactedwith this same reaction mixture but without any NTR or thioredoxin.

EXAMPLE 15 Wheat Bread Baking Studies

The baking tests were carried out by using a computer monitoredPANASONIC baking apparatus.

Composition of bread: Control: Flour*: 200 gm (dry) Water: 70%hydratation Salt (NaCl): 5.3 g Yeast: 4.8 g (Saccharomiyces cerevisiae,SafInstant) (dry yeast powder) *Flour samples were obtained from purebread wheat cultivars having contrasting baking quality (includinganimal feed grade and other grades having from poor to good bakingquality).Assays

The dough for the assays contained all the components of the controlplus as indicated varying amounts of the NADP Thioredoxin System (NTS)and/or the NADP generating System.

Experimental Conditions

-   -   Flour and salt are weighed and mixed    -   The volume of water needed to reach a hydratation of 70% was        put. into the baking pan.    -   The mixture of flour and salt was added to the water and the        baking program monitored by the computer was started. The        complete program lasted 3 hrs 9 min and 7 secs.    -   In the case of the assays, enzyme system components are added to        the water before the addition of the flour-salt mixture.    -   Yeast was added automatically after mixing for 20 min and 3        secs.

The program monitoring the Panasonic apparatus was:

Mixing Segments Duration Conditions Heating Mixing 00:00:03 T1 offMixing 00:05:00 T2 off Mixing 00:05:00 T1 off Rest 00:10:00 T0 offMixing 00:17:00 T2 off Mixing 00:07:00 T1 off Rest 00:30:00 T0 to reach32° C. Mixing 00:00:04 T1  32° C. Rest 01:15:00 T0  32° C. Baking00:14:00 T0 to reach 180° C. Baking 00:26:00 T0 180° C.

Mixing Conditions:

-   T0=no mixing (motor at rest)-   T1=normal mixing-   T2=alternately 3 second mixing, 3 second rest

Bread loaf volume was determined at the end of the baking, when breadloaves reached room temperature.

Cultivar THESEE Assay

The french wheat cultivar Thesee is classified as having goodbreadmaking quality. Table VI below sets forth the results of the assay.

TABLE VI Loaf Volume NADPH Relative (μmoles) NTR (μg) Th (μg) (cm3)Units Control 0 0 0 1690 100 Samples 6.0 30 60 1810 107 6.0 30 0 1725102 6.0 0 60 1720 102 6.0 0 0 1550 92 0 30 60 1800 107 *NADPH 30 60 162096 Generating syst. *NADPH 30 60 1630 96 Generating syst. plus ATP,glucose NTR and 6.0 9.4 20 1750 104 Th from yeast Volume Added NADP, 25mMolar 700 μl (17.5 μmoles) Glucose-6-phosphate, 25 mMolar 700 μl (17.5μmoles) Glucose-6-phosphate dehydrogenase 175 μl (8.75 μg) (50 μg/ml)ATP, 25 mMolar 700 μl (17.5 μmoles) Glucose, 25 mMolar 700 μl (17.5μmoles) *Composition of the NADPH generating system, ATP and glucose.

As shown in Table VI, an increased loaf volume was obtained when thecomplete NTS at concentrations of 6.0 μmoles NADPH, 30 μg NTR and 60 μgTh was used to bake loaves from 200 g of Thesee flour with the amountsand conditions described above in this Example. Unless otherwise stated,the NTR and thioredoxin (th) were from E. coli. No similar increaseoccurred when the generating system was used or when either NTR or Thwere omitted. Also no significant effect on loaf volume occurred whenamounts of the components in the system were about half or less thanhalf of the amounts of above.

Cultivar APOLLO Assay

This French wheat cultivar is classified as having poor breadmakingquality. The NTR and thioredoxin used in this assay were from E. coli.Table VII below sets forth the results of this assay using 200 gm ofApollo flour. Again unless otherwise stated the amounts and conditionsare those described above at the beginning of the Example.

TABLE VII Loaf Volume NADPH Relative (μmoles) NTR (μg) Th (μg) (cm3)Units Control 0 0 0 1400 100 Samples 6.0 30 60 1475 105 *NADPH 30 601530 109 Generating syst. plus ATP, glucose *NADPH 0 0 1430 102Generating syst. plus ATP, glucose *NADPH 6 0 1430 102 Generating syst.*NADPH 6 7 1440 103 Generating syst. *The composition of the generatingsystem, ATP and glucose is as in Table VI.Cultivar ARBON Assay

The French wheat cultivar Arbon is used for feed and is classified asnon suitable for breadmaking. Tables VIII and IX below show that animproved bread loaf volume can be obtained from Arbon using the NTS orNADPH and NTR with the dough components and conditions described at thebeginning of the Example. The amounts of NTR, thioredoxin, NADPH and theNADPH generating system components used in the assay are set forth inTables VIII and IX. The improvement in Arbon bread quality using thecomplete NTS as set forth in Table IX was clearly visible when comparedto the control.

TABLE VIII NADPH (μmoles) NTR (μg) Th (μg) Loaf Volume (cm3) Control 0 00 1350 Samples 0.1–0.6 3–4 3–4 up to 20% higher than thecontrol >2.0 >20 >20 less than the control

TABLE IX Loaf Volume Relative Treatment cm3 Units Complete NTS 1650 122minus Thioredoxin 1690 125 minus NTR 1520 113 minus Thioredoxin, NTR1540 114 minus NADPH 1440 107 minus NADPH, 1560 116 plus *NADPHgenerating system minus NTS (control) 1350 100 NADPH, 0.6 μmolesThioredoxin, 3.5 μg NTR, 3 μg *Generating System: 3.5 μmoles NADP 3.5μmoles glucose-6-phosphate 1.75 μg glucose-6-phosphate dehydrogenase

EXAMPLE 16 Triticale Bread Baking Study

Triticale is a wheat/rye hybrid and is generally used for chicken feed.It is more nutritious than wheat but is not generally consideredappropriate for breadmaking, especially in the more developed nations.The effect of the NTS system and variations thereof on loaves baked fromTriticale flour was consequently studied. Unless otherwise stated, thebaking conditions and dough ingredient were as described for wheat flourin Example 15. As shown in Table X there is an improvement in loafvolume when the triticale dough contained thioredoxin, NTR and the NADPHgenerating system in the amounts set forth in that Table. However, nocorresponding improvement was seen when the NTS (i.e., thioredoxin, NTRand NADPH) was used. An improvement in the texture of the bread, makingit more cohesive and stable, also occurred when NTR, Th and the NADPHgenerating system as set forth in Table X were used.

TABLE X Effect of the NADP/Thioredoxin System (NTS) on Loaves Baked fromTriticale Flour (cv. Juan) Loaf Volume Relative Treatment (cm3) UnitsComplete NTS 1230 94 minus NTS (control) 1310 100 minus NADPH, plus*NADPH 1390 106 NADPH, 0.6 μmoles Thioredoxin, 3.5 μg NTR, 3.0 μgGenerating System: 4.5 μmoles NADP 4.5 μmoles glucose-6-phosphate 4.5 μgglucose-6-phosphate dehydrogenase

EXAMPLE 17

The effect of the NADPH/thioredoxin system on flour from sorghum, cornand rice was also determined. The baking conditions were as describedfor wheat flour in Example 15. The amounts of the components of the NTSas used in this assay were as follows: 8 μmoles NADPH, 40.5 μg NTR and54 μg thioredoxin. Both the thioredoxin and NTR were from E. coli. Thebreads in this study containing the NTS, especially corn and sorghumexhibited improved texture and stability.

EXAMPLE 18 Reduction of Ethanol-Soluble and Myristate-Soluble StorageProteins from Triticale, Rye, Barley, Oat, Rice, Sorghum, Corn and Teff

Unless otherwise stated, the materials and methods used in this Exampleare according to those set forth above in the section titled “Reductionof Cereal Proteins, Materials and Methods.”

Triticale, Rye, Barley, Oat and Teff

The reactions were carried out in 30 mM Tris-HCl buffer, pH 7.9. Asindicated, 0.7 μg of NTR and 1 μg of thioredoxin from E. coli or 2 μg ofthioredoxin from yeast, as identified, were added to 70 μL of thisbuffer containing 1 mM NADPH and 25 to 30 μg of extracted storageprotein. The ethanol extracted storage proteins were obtained by using50 ml of 70% ethanol for every 10 gm of flour and extracting for 2 hr.In the case of teff, 200 mg of ground seeds were extracted. Themyristate extracted proteins were obtained by extracting 1 gm of flourwith 8 mg sodium myristate in 5 ml of distilled H₂O for 2 hrs. Thecombination of NADPH, NTR and thioredoxin is known as theNADP/thioredoxin system (NTS). As indicated, glutathione (GSH), 2.5 mM,was added as reductant in either the absence (GSH) or presence of 1.5 mMNADPH and 1.4 μg of spinach leaf glutathione reductase (GR/GSH/NADPH).After incubation for 20 min, 100 nmol of mBBr was added and the reactionwas continued for another 15 min. To stop the reaction and derivatizeexcess mBBr, 10 μL of 10% SDS and 10 μL of 100 mM 2-mercaptoethanol wereadded, and the samples were then applied to the gels. The procedure forSDS-polyacrylamide gel electrophoresis was as described by N. A.Crawford et al. (1989 Arch. Biochem. Biophys. 271:223–239).

Rice, Sorghum and Corn

The reactions were carried out in 30 mM Tris-HCl buffer, pH 7.9. Whenproteins were reduced by thioredoxin, the following were added to 70 μLof buffer: 1.2 mM NADPH, 10 to 30 μg of seed protein fraction, 0.5 Ig E.coli NTR and 1 ug E. coli thioredoxin. For reduction with glutathione,thioredoxin and NTR were replaced with 2.5 mM reduced glutathione and 1μg glutathione reductase (baker's yeast, Sigma Chemical Co.). Forreduction with dithiothreitol, NADPH, thioredoxin, and NTR were omittedand 0.5 mM dithiothreitol was added. In all cases, incubation time was20 min. Then 10 μl of a 10 mM mBBr solution was added and the reactioncontinued for an additional 15 min. To stop the reaction and derivatizeexcess mBBr, 10 μl of 10% SDS and 10 μl of 100 mM 2-mercaptoethanol wereadded and the samples applied to the gels. In each case, to obtain theextracted protein, 1 g ground seeds was extracted with 8 mg of sodiummyristate in 5 ml distilled water. With the exception of the initialredox state determination of the proteins, samples were extracted for 2hr at 22° C. and then centrifuged 20 min at 16,000 rpm prior to theaddition of the mBBr. With the initial redox state determination, themBBr was added under a nitrogen atmosphere along with the myristatefollowed by extraction.

Separate SDSS-polyacrylamide electrophoretic gels of the reductionstudies of myristate-extracted proteins from flour of oat, triticale,rye, barley and teff were prepared. A gel showing the extent ofthioredoxin linked buffer and ethanol-extracted proteins for teff wasalso prepared. In all of the oat, triticale, rye, barley, teff/myristateextractions studies, the flour was first extracted with buffer, 50 mMTris-HCl, pH 7.5 for 20 min and then with 70% ethanol for 2 hr. Inaddition, gels were prepared for the myristate-extracted proteins fromcorn, sorghum and rice. With corn, sorghum and rice, the ground seedswere extracted only with myristate. Therefore, with corn, sorghum andrice, the myristate extract represents total protein, whereas with oat,triticale, rye, barley and teff, the myristate extract represents onlythe glutenin-equivalent fractions since these flours had been previouslyextracted with buffer and ethanol. The results, depicted in the gels,show that the NTS is most effective, as compared to GSH or GSH/GR/NADPH,with myristate-extracted (glutenin-equivalent) proteins from oat,triticale, rye, barley and teff. The NTS is also most effective with thetotal proteins from rice. Reduced glutathione is more effective with thetotal proteins from corn and sorghum.

Conclusions from the Corn, Sorghum and Rice

In the first gel relating to the effect of NTS vs. glutathione reductaseon the reduction status of the myristate-extracted proteins, intreatment (1), extraction with myristate in the presence of mBBr wascarried out under a nitrogen atmosphere; in treatment (2), to themyristate extracted proteins mBBr was added without prior reduction ofthe proteins; in treatment (3), the myristate extracted proteins werereduced by the NADP/thioredoxin system (NTS); in treatment (4) themyristate extracted proteins were reduced by NADPH, glutathione andglutathione reductase. In the second gel relating to the in vivoreduction status and thioredoxin linked in vitro reduction of themyristate-extracted proteins, treatment (1) is like treatment (2) in thefirst gel; in treatment (2) the seeds were extracted with myristate inthe presence of mBBr under nitrogen; in treatment (3), seeds wereextracted with myristate and reduced by the NTS and then mBBr was added;and in treatment (4) conditions as in (3) except that proteins werereduced by DTT. Treatment (1) in the first gel and treatment (2) in thesecond gel showed the initial redox state of the proteins in the grains.For all three cereals, the proteins in the seed were highly reduced. Ifextracted in air, the proteins became oxidized especially the sorghumand rice. The oxidized proteins can be re-reduced, maximally with NTS inall cases. With rice, the reduction was relatively specific forthioredoxin; with corn, glutathione is as effective as thioredoxin andwith sorghum glutathione is slightly more effective than thioredoxin.Dithiothretol showed varying effectiveness as a reductant. Theseexperiments demonstrated that the storage proteins of these cereals areless specific than in the case of wheat and suggest that thioredoxinshould be tested both in the presence and absence of glutathione whenattempting to construct a dough network.

Gels were also prepared resulting from the reduction studies of wheatglutenins and gliadins, respectively, by a yeast NADP/thioredoxinsystem. The glutenins were obtained by using 50 ml of 0.1 M acetic acidfor every 10 gm of flour and extracting for 2 hr. The gliadins wereobtained by using 50 ml of 70% ethanol for every 10 gm of flour andextracting for 2 hr. The gels showed that the yeast system is highlyactive in reducing the two major groups of wheat storage proteins.

gels for the reduction of ethanol-extracted proteins from flour oftriticale, rye, oat and barley, respectively, were also prepared. Theresults showed that the NTS is most effective with the ethanol-extractedproteins from triticale, rye and oat. The ethanol-extracted barleyproteins were reduced in the control and thioredoxin or glutathione hadlittle effect.

EXAMPLE 19 Effect of Thioredoxin-linked Reduction on the Activity andStability of the Kunitz and Bowman-Birk Soybean Trypsin InhibitorProteins Materials and Methods

Plant Materials

Durum wheat (Triticum durun, Desf. cv. Monroe) was a kind gift of Dr. K.Kahn. Wheat germ was obtained from Sigma Chemical Co. (St. Louis, Mo.).

Chemicals and Enzymes

Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) were obtained from Bio-Rad Laboratories (Richmond, Calif.),and DTT was from Boehringer Mannheim Biochemicals (Indianapolis, Ind.).L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin(type XIII, T8640), subtilisin (type VIII: bacterial subtilisinCarbsberg, P5380), KTI (T9003), BBTI (T9777), azocasein, and otherchemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). E.coli thioredoxin and NTR were isolated from cells transformed tooverexpress each protein. The thioredoxin strain containing therecombinant plasmid, pFPI, was kindly provided by Dr. J.-P. Jacquot (deLa Motte-Guery et al., 1991). The NTR strain containing the recombinantplasmid, pPMR21, was kindly provided by Drs. Marjorie Russel and PeterModel (Russel and Model, 1988). The isolation procedures used for theseproteins were as described in those studies with the following changes:cells were broken in a Ribi cell fractionator at 25,000 psi and NTR waspurified as described by Florencio et al. (1988) without the red agarosestep. The E. coli thioredoxin and NTR were, respectively, 100% and 90%pure as determined by SDS-polyacrylamide gel electrophoresis. Wheatthioredoxin h was purified as previously described (Johnson et al.,1987).

Germination of Wheat Seeds

Wheat seeds were sterilized by steeping in 50% (v/v) of Generic Bleachfor 1 h at room temperature, followed by a thorough wash with distilledwater. The sterilized seeds were placed in a plastic Petri dish on twolayers of Whatman filter paper moistened with distilled water containing100 μg/ml of chloramphenicol. Germination was continued at roomtemperature in a dark chamber for up to 5 days.

Preparation of Wheat Proteases

The endosperm (10–15 g fresh weight) isolated from 5-day germinatedwheat seeds by excising the roots and shoots was extracted for 30minutes at 4° C. with 5 volumes of 200 mM sodium acetate, pH 4.6,containing 10 mM β-mercaptoenthanol. The homogenate was centrifuged for20 minutes at 48,000 g, 4° C. The pellet was discarded and thesupernatant fluid was fractionated with 30–70% ammonium sulfate. Thisfraction, which represented the protease preparation, was resuspended ina minimum volume of 20 mM sodium acetate, pH 4.6, containing 10 mMβ-mercaptoenthanol, and dialyzed against this buffer overnight at 4° C.When assayed with azocasein as substrate, the protease preparation hadan optimal pH of about 4.6 and was stable for at least one week at 4° C.

Reduction and Proteolytic Susceptibility of Trypsin Inhibitors

Unless indicated, the reduction of the trypsin inhibitors (0.4 mg/ml)was carried out in 0.1 ml of 20 mM sodium phosphate buffer, pH 7.9containing 10 mM EDTA at 30° C. for 2 hours. The concentrations ofthioredoxin, NTR, and NADPH were 0.024 mg/ml, 0.02 mg/ml, and 0.25 mM,respectively. With DTT as reductant, EDTA and components of theNADP/thioredoxin system were omitted. Following reduction, aliquots ofthe inhibitor mixture were withdrawn either for determination of trypsininhibitory activity or proteolytic susceptibility. In the subtilisintests, the inhibitor mixture (50 μl) was directly mixed with subtilisinand incubated at room temperature for 1 hour. With the wheat proteasepreparation, the pH of the inhibitor mixture (50 μl) was first adjustedto 4.7 by mixing with 35 μl of 200 mM sodium acetate, pH 4.6; 10 μl ofthe wheat protease preparation was then added and incubation wascontinued for 2 hours at 37° C. To stop digestion with subtilisin, 2 μlof 100 mM phenylmethylsulfonyl fluoride (PMSF) and 10 μl of 10% SDS wereadded to the digestion mixture. With the plant protease preparation,digestion was stopped by adding an equal volume of SDS sample buffer[0.125 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v)β-mercaptoethanol, and 0.02% (w/v) bromophenol blue]. Proteolyticproducts were analyzed by electrophoresis with 12% or 16% SDSpolyacrylamide slab gels (Laemmli, 1970). The dried slab gels werescanned with a laser densitometer (Pharmacia-LKB UltraScan XL) and thepeak area of the KTI or BBTI protein band was obtained by integrationwith a Pharmacia GelScan XL software program.

Assays

Thioredoxin and NTR were assayed as previously described by Florencio etal. (1988). Trypsin activity was measured in 50 mM Tris-HCl, pH 7.9, byfollowing the increase in absorbance at 253 Dm with N-benzoyl-L-arginineethyl ester as substrate (Mundy et al., 1984) or by the release of azodye into the trichloroacetic acid (TCA)-soluble fraction from azocaseinsubstrate (see below). For trypsin inhibition assays, trypsin (5 to 10μg) was preincubated with appropriate amounts of KTI or BBTI for 5minutes at room temperature in 50 mM Tris-HCl, pH 7.9 and proteolyticactivity was then determined. While the two substrates yielded similardata, results are presented with only one substrate.

Wheat protease activity was measured by following the release of azo dyeinto TCA solution from azocasein substrate at pH 4.7. Fifty μl of wheatprotease in a solution of 20 mM sodium acetate, pH 4.6, and 10 mMβ-mercaptoethanol were added to 50 μl of 200 mM sodium acetate, pH 4.6,and 100 μl of 2% azocasein (in 20 mM sodium phosphate, pH 7.0).Following 1-hour incubation at 37° C., 1 ml of 10% TCA was added and themixture was allowed to stand for 10 minutes at room temperature. Aftercentrifugation for 5 minutes in a microfuge (8000 g), 1 ml of thesupernatant solution was withdrawn and mixed with 1 ml of 1 N NaOH. Theabsorbance was read at 440 nm. Protein concentration was determined withBio-Rad reagent using bovine serum albumin as a standard (Bradford,1976).

Results

Trypsin Inhibitory Activity

The 20 kDa Kunitz and 8 kDa Bowman-Birk trypsin inhibitors of soybeancontain 2 and 7 disulfide groups, respectively (Birk, 1976; Wilson,1988). Although their physiological functions have not been established,the two types of inhibitors have been extensively investigated owing totheir wide distribution in legume seeds and their potential to causenutritional disorders, e.g., hypertrophy and associated malfunctions ofthe pancreas. As shown in Tables I and II and described in previousExamples, KTI and BBTI are reduced specifically by the NADP/thioredoxinsystem from either E. coli or plants. The reduced forms of glutathioneand glutaredoxin (a thiol protein capable of replacing thioredoxin incertain animal and bacterial systems, but not known to occur in plants(Holmgren, 1985)) were without effect.

To determine the consequence of reduction by thioredoxin, the trypsininhibitory activity of the oxidized and reduced forms of KTI and BBTIwas compared. As shown in Table XI, preincubation with theNADP/thioredoxin system (NTS) for 2 hours at 30° C. resulted in asubstantial loss of trypsin inhibitory activity (i.e., there was anincrease in trypsin activity relative to the uninhibited control). Morespecifically, the NADP/thioredoxin system effected a 3- and 6-foldincrease in trypsin activity for KTI and BBTI, respectively. Similarresults were obtained with DTT, a nonphysiological substitute forthioredoxin, and with thioredoxin reduced by lipoic acid, a naturallyoccurring dithiol. Extended incubation with DTT alone (overnight at roomtemperature) led to complete or almost complete inactivation of bothinhibitors (data not shown). Unlike DTT, lipoic acid did not reduce(inactivate) KTI and BBTI significantly in the absence of thioredoxin.

TABLE XI Changes in the Ability of Soybean Trypsin Inhibitors to InhibitTrypsin Following Reduction by the NADP/Thioredoxin System, DTT orReduced Lipoic Acid Relative Trypsin Activity* Treatment KTI BBTI Noinhibitor 100 100 Inhibitor Oxidized 17.0 11.5 Reduced by NTS¹ 55.6 70.6Reduced by DTT2 68.6 88.9 Reduced by LA/Trx h³ 40.5 87.8 *The specificactivity of the uninhibited control trypsin was 0.018 ΔA_(253 nm)/μg/minusing N-benzoyl-L-arginine ethyl ester as substrate. ¹Reduction by E.coli NTS (NADP/thioredoxin system) was conducted at 30° C. for 2 hours.²Reduction by DTT (1 mM) was conducted at 30° C. for 1 hour. ³Reductionby lipoic acid (LA, 0.4 mM) and wheat thioredoxin h (Trx h) wasconducted at 30° C. for 1 hour. In the presence of lipoic acid alone(0.4 mM), trypsin activity was 20.0% for KTI and 12.5% for BBTI.

Friedman and colleagues observed that heating soybean flour in thepresence of sulfur reductants (sodium sulfite, N-acetyl-L-cysteine,reduced glutathione, or L-cysteine) inactivated trypsin inhibitors,presumably as a result of the reduction or interchange of disulfidegroups with other proteins in soy flour (Friedman and Gumbmann, 1986;Friedman et al., 1982, 1984). Inactivation of the trypsin inhibitors bythese reductants improved the digestibility and nutritive value offlours in tested rats (Friedman and Gumbman, 1986). Taken together withthese earlier observations, the present findings demonstrate thatdisulfide bonds of both KTI and BBTI targeted by thioredoxin areimportant to maintenance of trypsin inhibitory activity.

Heat Stability

Protease inhibitor proteins are typically stable to inactivationtreatments such as heat. This stability is attributed, at least in part,to the cross-linking of disulfide bonds (Birk, 1976; Ryan, 1981). It isknown that breaking the disulfide bonds by reduction decreases heatstability (Friedman et al., 1982). The question arises as to whetherreduction by thioredoxin yields similar results.

The results as shown in TABLE XII provide a positive answer to thisquestion. When heated at 80° C. for 15 minutes, the thioredoxin-reducedform of KTI completely lost its ability to inhibit trypsin, whereas itsoxidized counterpart retained about half of the original activity (TableXII). Oxidized BBTI was even more stable, retaining the bulk of itstrypsin inhibitory activity after heating at 100° C. for 25 minutes.Nonetheless, as with KTI, the reduced form of BBTI was fully inactivatedby heat (Table XII). These results are consistent with priorobservations (i) that KTI and BBTI show increased sensitivity to heat onreduction; and (ii) that pure BBTI in solution is more heat-stable thanpure KTI in solution. The reverse is true for flour (i.e., KTI is moreheat-stable than BBTI (Friedman et al., 1982 and 1991; and DiPietro andLiener, 1989)).

TABLE XII Heat Stability of the Kunitz and Bowman-Birk TrypsinInhibitors: Oxidized and Following Reduction by the E. coliNADP/thioredoxin System Relative Trypsin Activity* Treatment KTI BBTI Noinhibitor 100 100 Inhibitor, unheated Oxidized 26.6 9.4 Reduced 76.482.4 Inhibitor, heated 15 min at 80° C. Oxidized 52.3 nd¹ Reduced 98.7nd  Inhibitor, heated 25 min at 100° C. Oxidized nd 17.2 Reduced nd 98.4*The specific activity of trypsin was 0.319 ΔA_(440 nm)/mg/min usingazocasein as substrate. The temperatures used for inactivation weredetermined in initial experiments designed to show the heat stability ofthe trypsin inhibitors under our conditions. ¹nd: not determined.Protease Susceptibility

To test whether the reduced forms of KTI and BBTI show decreasedstability to proteases other than trypsin, both the reduced and oxidizedforms of KTI and BBTI were incubated with a wheat protease preparationor with subtilisin and the proteolytic products were analyzed bySDS-PAGE. The extent of proteolysis was determined by measuring theabundance of intact protein on SDS gels by laser densitometer. Whentested with a protease preparation from 5-day germinated wheat seeds,the oxidized form of the Kunitz inhibitor was almost completelyresistant to digestion whereas the thioredoxin-reduced form wassusceptible to protease. As shown in Table XIII, about 80% of KTI wasdegraded in a reaction that depended on all components of theNADP/thioredoxin system (NTS). BBTI showed the same pattern except thatthe oxidized protein showed greater proteolytic susceptibility relativeto KTI. Similar effects were observed with both inhibitors when theplant protease preparation was replaced by subtilisin (data not shown).The nature of the proteolytic reaction was not investigated, but it isnoted that peptide products were not detected on SDS gels.

TABLE XIII Effect of Thioredoxin-linked Reduction on the Susceptibilityof Kunitz and Bowman-Birk Trypsin Inhibitors to Proteolysis by a PlantProtease Preparation¹ Relative Abundance² Treatment KTI BBTI No protease100 100 Protease No reduction system 97.9 67.2 E. coli NTS³ 22.1 16.0NTS minus thioredoxin 90.2  nd⁴ NTS minus NADPH 97.7 nd NTS minus NTR97.9 nd ¹Following reduction by E. coli thioredoxin system at 30° C. for2 hours, pH was adjusted to 4.7 by addition of 200 mM sodium acetate, pH4.6. Wheat protease preparation was then added and incubated at 37° C.for 2 hours, followed by SDS-PAGE analyses. ²Determined by laserdensitometer. ³NTS: NADP/thioredoxin system. ⁴nd: not determined.

This Example shows that reduction by thioredoxin, or dithiothreitol(DTT), leads to inactivation of both proteins and to an increase intheir heat and protease susceptibility. The results indicate thatthioredoxin-linked reduction of the inhibitor proteins is relevant bothto their industrial processing and to seed germination.

These results confirm the conclusion that disulfide bonds are essentialfor the trypsin inhibitory activity of KTI and BBTI (Birk, 1985;Friedman and Gumbmann, 1986; Friedman et al., 1982,1984). These studiesalso show that reduction (inactivation) can take place underphysiological conditions (i.e., at low temperature with NADPH-reducedthioredoxin). The ability to inactivate the trypsin inhibitors at lowertemperatures provides a potential method for full inactivation of bothtrypsin inhibitors, thereby improving the quality of soybean productsand saving energy. The need for a method for the complete inactivationof KTI is significant since 20% of its activity is consistently retainedin soy flour under conditions in which BBTI is fully inactivated(Friedman et al., 1991).

The present results also add new information on the proteasesusceptibility of KTI and BBTI. Their increase in proteasesusceptibility following reduction suggests that, if exposed to theprotease inhibitors during seed germination, the NADP/thioredoxin systemcould serve as a mechanism by which the inhibitor proteins are modified(inactivated) and eventually degraded (Baumgartner and Chrispeels, 1976;Chrispeels and Baumgartner, 1978; Orf et al., 1977; Wilson, 1988;Yoshikawa et al., 1979). As stated previously, there is evidence thatthe NADP-thioredoxin system plays a similar role in mobilizing proteinsduring the germination of wheat seeds.

EXAMPLE 20 Reduction of Castor Seed 2S Albumin Protein by Thioredoxin

The results of the following study of sulfhydryl agents to reduce the 2Sprotein from castor seed (Sharief and Li, 1982; Youle and Huang, 1978)shows that thioredoxin actively reduces intramolecular disulfides of the2S large subunit but not the intermolecular disulfides joining the twosubunits.

Materials and Methods

Materials

Seeds of castor (Ricinus communis L. var Hale) were obtained fromBothwell Enterprises, Plainview, Tex.). Biochemicals were obtained fromSigma Chemical Co. (St. Louis, Mo.). E. coli thioredoxin and NTR wereisolated from cells transformed to overexpress each protein. Thethioredoxin strain containing the recombinant plasmid pFPI, was kindlyprovided by Dr. J.-P. Jacquot (de La Mott-Guery et al. 1991). The straincontaining the recombinant plasmid, pPMR21, was kindly provided by Drs.Marjorie Russel and Peter Model (Russel and Model, 1988). Thioredoxinand NTR were purified by the respective procedures of de La Mott-Gueryet al. (1991) and Florencio et al. (1988). Reagents forSDS-polyacrylamide gel electrophoresis were purchased from Bio-RadLaboratories (Richmond, Calif.). Monobromobimane (mBBr) or Thiolite wasobtained from Calbiochem (San Diego, Calif.). Other chemicals wereobtained from commercial sources and were of the highest qualityavailable. NADP-malate dehydrogenase and fructose-1,6-bisphosphatasewere purified from leaves of corn (Jacquot et al. 1981) and spinach(Nishizawa et al. 1982), respectively. Thioredoxin h was isolated fromwheat seeds by following the procedure devised for the spinach protein(Florencio et al. 1988). Glutathione reductase was prepared from spinachleaves (Florencio et al. 1988).

Isolation of Protein Bodies

Protein bodies were isolated by a nonaqueous method (Yatsu and Jacks,1968). Shelled dry castor seeds, 15 g, were blended with 40 ml ofglycerol for 30 sec in a Waring blender. The mixture was filteredthrough four layers of nylon cloth. The crude extract was centrifuged at272×g for 5 min in a Beckman J2-21M centrifuge using a JS-20 rotor.After centrifugation, the supernatant fraction was collected andcentrifuged 20 min at 41,400×g. The pellet, containing the proteinbodies, was resuspended in 10 ml glycerol and centrifuged as before(41,400×g for 20 min) collecting the pellet. This washing step wasrepeated twice. The soluble (“matrix”) fraction was obtained byextracting the pellet with 3 ml of 100 mM Tris-HCl buffer (pH 8.5). Theremaining insoluble (“crystalloid”) fraction, collected bycentrifugation as before, was extracted with 3 ml of 6M urea in 100 mMTris-HCl buffer (pH 8.5).

2S Protein Purification Procedure

The 2S protein was prepared by a modification of the method of Tully andBeevers (1976). The matrix protein fraction was applied to aDEAE-cellulose (DE-52) column equilibrated with 5 mM Tris-HCl buffer, pH8.5 (Buffer A) and eluted with a 0 to 300 mM NaCl gradient in buffer A.Fractions containing the 2S protein were pooled and concentrated byfreeze drying. The concentrated fraction was applied to a Pharmacia FPLCSuperose-12 (HR 10/30) column equilibrated with buffer A containing 150mM NaCl. The fraction containing, 2S protein from the Superose-12 columnwas applied to an FPLC Mono Q HR 5/5 column equilibrated with buffer A.The column was eluted sequentially with 3 ml of buffer A, 20 ml of alinear gradient of 0 to 300 mM NaCl in buffer A and finally with bufferA containing 1 M NaCl. The 2S protein purified by this method was freeof contaminants in SDS polyacrylamide gels stained with Coomassie blue(Kobrehel et al., 1991).

Analytical Methods

Reduction of proteins was monitored by the monobromobimane (mBBr)/SDSpolyacrylamide gel electrophoresis procedure of Crawford et al. (1989).Labeled proteins were quantified as described previously in the“Reduction of Cereal Proteins, Materials and Methods” section. Proteinwas determined by the method of Bradford (1976).

Enzyme Assays/Reduction Experiments

The Wada et al., 1981 protocol was used for assaying NADP-malatedehydrogenase and fructose 1,6 bisphosphatase in the presence ofthioredoxin and 2S protein. Assays were conducted under conditions inwhich the amount of added thioredoxin was sufficient to reduce thecastor 2S protein but insufficient to activate the target enzymeappreciably. All assays were at 25° C. Unless otherwise indicated, thethioredoxin and NTR used were from E. coli. The 2S protein was monitoredduring purification by mBBr/SDS-polyacrylamide gel electrophoresisfollowing its reduction by dithiothreitol and E. coli thioredoxin(Crawford et al., 1989; Kobrehel et al., 1991).

The reduction of the matrix and crystalloid proteins from castor seedwere determined by the mBBr/SDS-polyacrylamide gel electrophoresisprocedure. The lanes for the gels (not shown) were as follows, 1 and 7,Control: no addition; 2 and 8, GSH/GR/NADPH: reduced glutathione,glutathione reductase (from spinach leaves) and NADPH; 3 and 9, NGS:NADPH, reduced glutathione, glutathione reductase (from spinach leaves)and glutaredoxin from E. coli; 4 and 10, NTS: NADPH, NTR, andthioredoxin (both proteins from E. coli); 5 and 11, NADPH; 6 and 12,NADPH and E. coli NTR. Reactions were carried out in 100 mM Tris-HClbuffer, pH 7.8. As indicated, 0.7 μg NTR and 1 μg thioredoxin were addedto 70 μl of this buffer containing 1 mM NADPH and target protein: 8 μgmatrix protein for treatments 1–6 and 10 μg crystalloid protein fortreatments 7–12. Assays with glutathione were performed similarly, butat a final concentration of 2 mM, 1.4 μg glutathione reductase, 1 μgglutaredoxin, and 1.5 mM NADPH. Reaction time was 20 min.

The mBBr/SDS-Page technique was also used to determine the specificityof thioredoxin for reducing the disulfide bonds of castor seed 2Sprotein. The lanes for the gel (not shown) were as follows, (1) Control(no addition); (2) Control+NTS (same conditions as with the matrix andcrystalloid proteins); (3) Control (heated 3 min at 100° C.); (4)Control+2 mM DTT (heated 3 min at 100° C.). The samples containing 5 μg2S protein and the indicated additions were incubated for 20 min in 30mM Tris-HCl (pH 7.8). mBBr, 80 nmol, was then added and the reactioncontinued for another 15 min prior to analysis by the mBBr/SDSpolyacrylamide gel electrophoresis procedure.

Results

The castor storage proteins, which are retained within a protein bodyduring seed maturation, can be separated into two fractions on the basisof their solubility. The more soluble proteins are housed in the proteinbody outer section (“matrix”) and the less soluble in the inner(“crystalloid”). In the current study, the matrix and crystalloidcomponents were isolated to determine their ability to undergo reductionby cellular thiols, viz., glutathione, glutaredoxin and thioredoxin.Glutaredoxin, a 12 kDa protein with a catalytically active thiol group,can replace thioredoxin in certain enzymic reactions of bacteria andanimals (Holmgren et al. 1985) but is not known to occur in plants.

The results showed that, while a number of storage proteins of castorseed were reduced by the thiols tested, only a low molecular weightprotein, corresponding to the large subunit of the 2S protein of thematrix, showed strict specificity for thioredoxin. Certain highermolecular weight proteins of the crystalloid fraction underwentreduction, but in those cases there was little difference betweenglutaredoxin and thioredoxin. The castor seed 2S large subunit thusappeared to resemble cystine-containing proteins previously discussed inundergoing thioredoxin-specific reduction. These experiments weredesigned to confirm this specificity and to elucidate certain propertiesof the reduced protein. As expected, owing to lack of disulfide groups,the 2S small subunit showed essentially no reaction with mBBr with anyof the reductants tested.

When its fluorescent band was monitored by laser densitometry, thereduction of the castor seed 2S large subunit was found to depend on allcomponents of the NADP/thioredoxin system (NADPH, NTR and thioredoxin)(Table XIV). As for other thioredoxin-linked proteins (includingchloroplast enzymes), the thioredoxin active in reduction of the 2Slarge subunit could be reduced either chemically with dithiothreitol(DTT) or enzymatically with NADPH and NTR. The extent of reduction bythe NADP thioredoxin system, DTT alone, and DTT+thioredoxin was 84%, 67%and 90%, respectively, after 20 min at 25° C. Similar, though generallyextensive reduction was observed with the disulfide proteins discussedabove (Johnson et al. 1987). As with the other seed proteins, nativewheat thioredoxin h and E. coli thioredoxins could be usedinterchangeably in the reduction of the 2S protein by DTT (data notshown).

TABLE XIV

Extent of reduction of the castor castor seed 2S protein by differentsulfhydryl reductants. Reaction conditions as with the matrix andcrytalloid protein determination. A reduction of 100% corresponds tothat obtained when the 2S protein was heated for 3 min in the presenceof 2% SDS and 2.5% (3-mercaptoethanol. NTS: NADPH, NTR, and thioredoxin(both proteins from E. coli); GSH/GR/NADPH: reduced glutathione,glutathione reductase (from spinach leaves) and NADPH; NGS: NADPH,reduced glutathione, glutathione reductase (from spinach leaves) andglutaredoxin (from E. coli).

Relative Treatment Reduction (%) Control 0 NADP/thioredoxin system,complete 84 NADP minus thioredoxin 0 NADP minus NADPH 0 NADP minus NTR 0Reduced glutathione 0 NADP/glutaredoxin system, complete 0 DTT 67 DTT +thioredoxin 90

The capability of thioredoxin to reduce the castor seed 2S protein wasalso evident in enzyme activation assays. Here, the protein targeted bythioredoxin (in this case 2S) is used to activate a thioredoxin-linkedenzyme of chloroplasts, NADP-malate dehydrogenase or fructose1,6bisphosphatase. As with most of the proteins examined so far, the 2Sprotein more effectively activated NADP-malate dehydrogenase and showedlittle activity with the fructose bisphosphatase (2.6 vs. 0.0nmoles/min/mg protein).

The castor seed 2S protein contains inter-as well as intramoleculardisulfides. The 2S protein thus provides an opportunity to determine thespecificity of thioredoxin for these two types of bonds. To this end,the castor seed 2S protein was reduced (i) enzymically with theNADP/thioredoxin system at room temperature, and (ii) chemically withDTT at 100° C. Following reaction with mBBr the reduced proteins wereanalyzed by SDS-polyacrylamide gel electrophoresis carried out withoutadditional sulfhydryl agent. The results indicate that while thioredoxinactively reduced intramolecular disulfides, it was much less effectivewith intermolecular disulfides.

The present results extend the role of thioredoxin to the reduction ofthe 2S protein of castor seed, an oil producing plant. Thioredoxinspecifically reduced the intramolecular disulfides of the large subunitof the 2S protein and showed little activity for the intermoleculardisulfides joining the large and small subunits. Based on the resultswith the trypsin inhibitors of soybean, it is clear that reduction ofintramolecular disulfides by thioredoxin markedly increases thesusceptibility of disulfide proteins to proteolysis (Jiao et al. 1992a).It, however, remains to be seen whether reduction of the 2S proteintakes place prior to its proteolytic degradation (Youle and Huang, 1978)as appears to be the case for the major storage proteins of wheat. Arelated question raised by this work is whether the 2S protein ofcastor, as well as other oil producing plants such as brazil nut(Altenbach et al., 1987; Ampe et al., 1986), has a function in additionto that of a storage protein.

EXAMPLE 21 Thioredoxin-Dependent Deinhibition of Pullulanase of Cerealsby Inactivation of a Specific Inhibitor Protein

Assay of Pullulanase

1. Standard Curve of Maltotriose:

A series of concentrations of maltotriose (0 to 2 mg) in 0.1 to 0.2 mlwater or buffer were made in microfuge tubes. To this was added 0.2 mlof dinitrosalicylic acid (DA) reagent (mix 1 g of DA, 30 g of sodiumpotassium tartrate, and 20 ml of 2N NaOH with water to final volume of100 ml). The reagents were dissolved in a warm water bath. The mixturewas heated at 100° C. for 5 min and cooled down in a water bath (roomtemperature). Each sample was transferred to a glass tube that contained2 ml of water. Read A₄₉₃ vs water. ΔA₄₉₃ [A493 of sample containingmaltotriose was subtracted from A₄₉₃ of the blank (no maltotriose)] wasplotted against maltotriose concentrations.

2. Pullulanase Activity Assay:

Pullulanase activity is measured as the release of reducing sugar fromthe substrate pullulan. Typically 10–100 μl of pullulanase sample (in 20mM Tris-HCl, pH 7.5, or in 5–20 acetate-NA, pH 4.6) was mixed with25–100 μl of 200 mM Acetate-NA, pH 5.5 (this buffer serves to bringfinal pH of the assay to 5.5) and 10–20 μl of 2% (w/v) pullulan. Themixture was incubated at 37° C. for 30 to 120 min, depending on theactivity of pullulanase. The reaction was stopped by adding 200 μl of DAreagent. Reducing sugar was then determined as above.

Note:

-   -   1. When a crude extract of pullulanase obtained by the dialysis        of crude extracts or pullulanase obtained from a dialyzed 30–60%        ammonium sulfate fraction is used as a pullulanase source, it        must be thoroughly dialysed before assay because there are        reduced sugars in the crude extract. In other words the        backround of crude pullulanase samples from dialysed crude        extracts or a dialysed 30–60% ammonium sulfate fraction is very        high. In this case, the blank is made as follows: 200 μl of DA        reagent are added first, followed by the addition of enzyme        sample, pullulan and buffer.    -   2. When final concentrations of DTT (or β-mercaptoethanol (MET)        or GSH) are higher than 2 mM in the assay mixture, the OD₄₉₃        values will be greater than those of the minus-DTT (MET, GSH)        samples. DTT (MET, GSH) should be added to the blank, samples        without DTT during assay at the end of the reaction. Care should        be taken to make sure the final concentration of DTT in the        assay mixture is below 2 mM.        Purification of Pullulanase Inhibitor Extraction and Ammonium        Sulfate Fractionation

200 g of barley malt was ground to fine powder with an electric coffeegrinder and extracted with 600 ml of 5% (w/v) NaCl for 3 h at 30° C.Following centrifugation at 30,000 g and at 4° C. for 25 min, thesupernatant was fractionated by precipitation with solid ammoniumsulfate. Proteins precipitated between 30% and 60% saturated ammoniumsulfate were dissolved in a minimum volume of 20 mM Tris HCl, pH 7.5,and dialyzed against this buffer at 4° C. overnight.

DE52 Chromatography

The dialyzed sample was centrifuged to remove insoluble materials andthe supernatant adjusted to pH 4.6 with 2N formic acid. After pelletingthe acid-denatured protein, the supernatant was readjusted to pH 7.5with NH₄OH and loaded onto a DE52 column (2.5×26 cm) equilibrated with20 mM Tris-HCl, pH 7.5. Following wash with 80 ml of the above buffer,the column was eluted with a linear 0–500 mM Tris-HCl, pH 7.5. Fractionsof 6.7 ml were collected. Pullulanase was eluted at about 325 mM NaCland its inhibitor came off at about 100 mM NaCl. Pullulanase was furtherpurified through CM32 (20 mM sodium acetate, pH 4.6) and Sephacryl-200HR (30 mM Tris-HCl, pH 7.5, containing 200 mM NaCl and 1 mM EDTA)chromatography. Pullulanase inhibitor protein was purified as describedbelow.

CM32 Chromatography

The pullulanase inhibitor sample (about 90 ml) from the DE52 step wasplaced in a 150-ml flask and incubated at 70° C. water-bath for 20 min.Following centrifugation, the clarified sample was then adjusted to pH4.6 with 2N formic acid and dialyzed against 20 mM sodium acetate, pH4.6. The precipitate formed during dialysis was removed bycentrifugation and the supernatant was chromatographed on a CM32 column(2.5×6 cm) equilibrated with 20 mM sodium acetate, pH 4.6. Proteins wereeluted with a linear 0–0.4 M NaCl in 200 ml of 20 mM sodium acetate, pH4.6. Fractions (5.0 ml/fraction) containing pullulanase inhibitoryactivity were pooled, dialyzed, and rechromatographed on a CM32 column(2.5×6 cm) with a linear 0.2–1 M NaCl gradient in 200 ml of 20 mM sodiumacetate, pH 4.0.

Sephadex G-75 Filtration

Pullulanase inhibitor fractions from the second CM32 chromatography stepwere concentrated in a dialysis bag against solid sucrose and thenseparated by a Sephadex G-75 column (2.5×85 cm) equilibrated with 30 mMTris-HCl, pH 7.5, containing 200 mM Na Cl and 1 mM EDTA. Fractions (3.6ml/fraction) showing pullulanase inhibitory activity were pooled,concentrated by dialysis against solid sucrose, and then dialysedagainst 10 mM Tris-HCl, pH 7.5.

Identification and Purification of Pullulanase Inhibitor

During gemination, starch is converted to glucose by α-, β-amylases, andpullulanase (also called debranching enzyme, R-enzyme). While extensivestudies have been conducted for the regulation of amylases, little isknown about the regulation of pullulanase in seeds. Yamada (Yamada, J.(1981) Carbohydrate Research 90:153–157) reported that incubation ofcereal flours with reductants (e.g., DTT) or proteases (e.g., trypsin)led to an activation of pullulanase activity, suggesting that reductionor proteolysis might be a mechanism by which pullulanase is activatedduring germination. Like in rice flour, pullulanase extracts fromgerminated wheat seeds or from barley malt showed lower activity, andwere activated 3 to 5-fold by preincubation with DTT for 20 to 30 min.However, following purification of the crude extract (a dialysate of30–60% ammonium sulfate fraction) by anion or cation exchangechromatography, the total pullulanase activity increased 2 to 3-foldover that of the sample applied to the column when assayed withoutpreincubation with DTT, and DTT has no or little effect on pullulanase.

One possibility was that pullulanase might be activated by proteolysisduring the process of purification, since germinated wheat seeds orbarley malt show high protease activity. If this was the case, additionof protease inhibitor cocktail would prevent pullulanase activationduring purification. In contrast to this point, many experiments withprotease inhibitors failed to prove this. Another possibility was thatthere is an inhibitor that is precipitated by ammonium sulfate andinhibits pullulanase. The role of DTT is to reduce and thus inactivatethis protein inhibitor, leading to activation of pullulanase. Along thisline, the 30–60% ammonium sulfate fraction from barley malt was appliedto a DE52 column (2.5×26 cm) equilibrated with 20 mM Tris-CH1, pH 7.5.Following elution with a linear salt gradient, “deinhibited”(“activated”) pullulanase was identified as a protein peak coming off atabout 325 mM NaCl (from fraction numbers 44 to 60). Assay of pullulanaseactivity in the preincubation mixture consisting of 50 μl of the peakpullulanase activity fraction (fraction number 45) with 50 μl of otherprotein fracitons indicated that a protein peak that showed pullulanaseinhibitory activity was eluted from the DE52 column by about 100 mM NaClbetween fraction numbers 8 to 25.

The pullulanase inhibitor sample was further purified by two consecutivecation exchange chromatography steps with CM32 at pH 4.6 and 4.0, andfiltration with Sephdex G-75.

Properties of Pullulanase Inhibitor

Preliminary experiments showed that pullulanase inhibitor protein isresistant to treatment of 70° C. for 10 min and pH 4.0. Based on theprofile of Sephadex G-75 gel filtration and SDS-PAGE, pullulanaseinhibitor has a molecular weight between 8 to 15 kDa±2 kDa. The studyfurther showed that the protein contains thioredoxin-reducible (S—S)bonds.

These studies, as shown in Table XV, found that the ubiquitous dithiolprotein, thioredoxin, serves as a specific reductant for a previouslyunknown disulfide-containing protein that inhibits pullulanase of barleyand wheat endosperm.

TABLE XV Activity Change in Pullulanase Inhibitor Protein FollowingReduction by NADP/Thioredoxin System Relative Pullulanase TreatmentActivity No inhibitor 100 Inhibitor Oxidized 30.1 Reduced by DTT 46.1Reduced by E. coli Trx/DTT 95.1 Reduced by E. coli NTS 40.4 Reduced byGSH/NADPH/GR 33.6

Reduction of the inhibitor protein eliminated its ability to inhibitpullulanase, thereby rendering the pullulanase enzyme active. Thesestudies as shown in Table XV illustrate that it is possible to renderthe pullulanase enzyme active with a physiological system consisting ofNADPH, NADP-thioredoxin reductase (NTR) and thioredoxin (theNADP/thioredoxin system) as well as with thioredoxin (Trx) anddithiothreitol. These findings also elucidate how reductive activationof pullulanase takes place (i.e., that a specific (previusly unknown)inhibitor is reduced and thereby inactivated, so that the enzyme becomesactive). The thioredoxin active in this reaction can be obtained fromseveral sources such as E. coli or seed endosperm (thioredoxin h). Therole of thioredoxin in reductively inactivating the inhibitor protein(I) and deinhibiting the pullulanase enzyme (E) is given in Equations 1and 2.

$\begin{matrix}{{{Thioredoxin}_{oxidized} + {NADPH}}\mspace{14mu}\overset{\text{~~~NTR}\;}{arrow}\mspace{14mu}{{Thioredoxin}_{oxidized} + {NADP}}} & (1) \\ {{Thioredoxin}_{oxidized} + \lbrack {E_{inactive}\text{:}I_{oxidized}} \rbrack}\mspace{14mu}arrow\mspace{14mu}{{Thioredoxin}_{oxidized} + E_{active} + I_{reduced}}  & (2)\end{matrix}$

In summary, the crude endosperm extracts were fractionated by columnchromatography procedures. These steps served to separate the proteininhibitor from the pululanase enzyme. The inhibitor protein was thenhighly purified by several steps. By use of the mBBr/SDS-PAGE procedure,it was determined that disulfide group(s) of the new protein arespecifically reduced by thioredoxin and that the reduced protein losesits ability to inhibit pullulanase. Like certain other disulfideproteins of seeds (e.g., the Kunitz and Bowman-Birk trypsin inhibitorsof soybean), the pullulanase inhibitor protein showed the capability toactivate chloroplast NADP-malate dehydrogenase. In these experiments,dithiothreitol was used to reduce thioredoxin, which in turn reducedinhibitor and thereby activated the dehydrogenase enzyme.

EXAMPLE 22 Engineering of Yeast Cells to Overexpress Thioredoxin andNADP-Thioredoxin Reductase

The two Saccharomyces cerevisiae thioredoxin genes (Muller, E. G. D.(1991), J. Biol. Chem. 266:9194–9202), TRX1 and TRX2, are cloned in highcopy number episomal vectors, an example of which is YEp24, under thecontrol of strong constitutive promoter elements, examples of which arethe glycolytic promoters for the glyceraldehyde-3-P dehydrogenase,enolase, or phosphoglycerate kinase genes. Recombinant constructs areassessed for the overexpression of thioredoxin by quantitative Westernblotting methods using an antithioredoxin rabbit antiserum (Muller, E.G. D. et al. (1989), J. Biol. Chem. 264:4008–4014), to select theoptimal combination of thioredoxin genes and promoter elements. Thecells with the optimal thioredoxin overexpression system are used as asource of thioredoxin for dough improvement.

The NADP-thioredoxin reductase gene is cloned by preparing anoligonucleotide probe deduced from its amino terminal sequence. Theenzyme is prepared from yeast cells by following a modification of theprocedure devised for spinach leaves (Florencio, F. J. et al. (1988),Arch. Biochem. Biophys. 266:496–507). The amino terminus of the purereductase enzyme is determined by microsequencing by automated Exmandegradation with an Applied Biosystems gas-phase protein sequencer. Onthe basis of this sequence, and relying on codon usage in yeast, a20-base 24-bold degenerate DNA probe is prepared. The probe ishybridized to isolated yeast DNA cleaved with EcoRI and PstI by Southernblot analysis. The most actively region is extracted from the agarosegels and introduced into a pUC19 plasmid vector (Szekeres, M. et al.(1991), J. Bacteriol. 173:1821–1823). Following transformation,plasmid-containing E. coli colonies are screened by colony hybridizationusing the labeled oligonucleotide probe (Vogeli, G. et al. (1987),Methods Enzymol. 152:407–415). The clone is identified as carrying thegene for NADP-thioredoxin reductase by sequencing the DNA as given inSzekeres et al. above. Once identified, the NADP-thioredoxin reductasegene is overexpressed in yeast as described above for the TRX1 and TRX2yeast thioredoxin genes. The yeast cells which overexpressNADP-thioredoxin reductase are used as a source of reductase to improvedough quality.

EXAMPLE 23 Improvement in Dough Quality Using Genetically EngineeredYeast Cells

Saccharoiznyces cerevisiae cells engineered to overexpress the two yeastthioredoxins and the yeast NADP-thioredoxin reductase as set forth inExample 23 are lysed by an established procedure such as sonication andthen freeze dried. The dried cells from the cultures overexpressingthioredoxin and NADP-thioredoxin reductase are combined and then used tosupplement flour to improve its dough quality. Two-tenths gram of thecombined lysed dried cells are added together with about 300 to about500 nanomoles NADPH to 1 M Tris-HCl buffer, pH 7.9, to give 5.25 ml of30 mM Tris-HCl. The reaction is carried out in a microfarinograph at 30°C. as described in Example 14. An improvement in dough quality isobserved which is similar to the improvement shown in Example 14.

EXAMPLE 24 Improvement of Gluten

The positive effects of the NADP/thioredoxin system on dough qualitypresents the option of applying this system to flour in the preparationof gluten. The purpose is to alter the yield and the properties ofgluten, thereby enhancing its technological value: (1) by obtainingstronger glutens (increased elasticity, improved extensibility); (2) byincreasing gluten yield by capturing soluble proteins, reduced by theNADP-thioredoxin system, in the protein network, thereby preventing themfrom being washed out during the production of gluten. In this procedure(using 10 g flour), 0.2 μg E. coli thioredoxin, 0.1 μg E. coliNADP-thioredoxin reductase and 300 to 500 nanomoles NADPH are addedtogether with 1 M Tris-HCl, pH 7.9, buffer to give 5.25 ml of 30 mMTris-HCl. The gluten is made at room temperature according to the commonlixiviation method. The yield of the gluten is determined by weight andthe strength of the gluten is determined by the classical manual stretchmethod. The gluten product which are obtained by this treatment with theNADP/thioredoxin system is used as an additive in flour or other grain.

EXAMPLE 25 Method of Producing Dough from a Non-wheat or Rye Flour

For this test (using 10 gm of milled flour from corn, rice or sorghum),0.2 μg E. coli thioredoxin, 0. 1 μg E. coli NADP-thioredoxin reductaseand 500 nanomoles NADPH are added together with 1 M Tris-HCl, pH 7.9,buffer to give 5.25 ml of 30 mM Tris-HCl. The reaction is carried out bymixing the 10 gm of milled flour with the enzyme system in amicro-farinograph at 30° C. The farinograph measurements show wheat-likedough characteristics by the added NADP-thioredoxin system. In thecontrols without the enzyme system, no microfarinograph reading ispossible because the mixture fails to form a dough. The dough which isformed is persistent and its consistency is maintained throughout therun. The end product is similar to the network formed in dough derivedfrom wheat.

Reduction of Animal Toxins

The invention provides a method for chemically reducing toxicity causingproteins contained in bee, scorpion and snake venome and therebyaltering the biological activity of the venoms well as reducing thetoxicity of animal toxins specifically snake neurotoxins by means ofthiol redox (SH) agents namely a reduced thioredoxin, reduced lipoicacid in the presence of a thioredoxin or DTT. The reduction of thethioredoxin occurs preferrably via the NADP-thioredoxin system (NTS). Asstated previously, the NTS comprises NADPH, NADP-thioredoxin reductase(NTR) and a thioredoxin.

The term “thiol Redox agent” has been used sometimes in the literatureto denote both an agent in the nonreduced state and also in the reducedor sulfhydryl (SH) state. As defined herein the term “thiol redox (SH)agent” means a reduced thiol redox protein or synthetically preparedagent such as DTT.

The reduction of the neurotoxin may take place in a medium that isliquid such as blood, lymph or a buffer, etc. or in a medium that issolid such as cells or other living tissue. As used herein the term“liquid” by itself does not refer to a biological fluid present in anindividual.

Presumably the proficiency of the thiol redox (SH) agents to inactivatethe venom in vitro and to detoxify the venom in individuals depends uponthe ability of the agents of the invention to reduce the intramoleculardisulfide bonds in these toxicity causing venom components.

All snake neurotoxins, both presynaptic and postsynaptic can be reducedand at least partially inactivated in vitro by the thiol redox (SH)agents of the invention. Snake neurotoxins inactivated in vitroaccording to the invention are useful as antigens in the preparation ofantivenoms. The neurotoxins are inactivated preferrably by incubationwith a thiol redox (SH) agent in an appropriate buffer. The preferredbuffer is Tris-HCl buffer but other buffers such as phosphate buffer maybe used. The preferred thiol redox (SH) agent is a reduced thioredoxin.

Effective amounts for inactivating snake neurotoxins range from about0.1 ttg to 5.0 μg, preferrably about 0.5 μg to 1.0 μg, of a reducedthioredoxin; from about 1 nanomole to 20 nanomoles, preferrably from 5nanomoles to 15 nanomoles, of reduced lipoic acid in the presence ofabout 1.0 μg of a thioredoxin and from about 10 nanomoles to 200nanomoles, preferrably 50 nanomoles to 100 nanomoles, of reduced DTT(preferrably in the presence of about 1.0 μg of a thioredoxin) for every10 μg of snake neurotoxin in a volume of 100 μl.

The effective amounts for inactivating a snake neurotoxin using thecomponents in the NADP-thioredoxin system range from about 0.1 μg to 5.0μg, preferrably about 0.5 μg to 1.0 μg, of thioredoxin; from about 0.1μg to 2.0 μg, preferrably from 0.2 μg to 1.0 μg, of NTR and from about0.05 micromoles to 0.5 micromoles, preferrably about 0. 1 micromoles to0.25 micromoles, of NADPH for every 10 μg of snake neurotoxin in avolume of 100 μl.

Upon inactivation the buffer containing the inactivated neurotoxin andthiol redox (SH) agent, etc. may be injected into an animal such as ahorse to produce an antivenom or prior to injection it may be furthertreated with heat or formaldehyde.

The thiol redox (SH) agents of the invention may also be used to treatindividuals who are suffering the effects of neurotoxicity caused by avenomous snake bite. The preferred method of administering the reducedthiol redox (SH) agent to the individual is by multiple subcutaneousinjections around the snake bite.

Of course the correct amount of a thiol redox (SH) agent used todetoxify a neurotoxin in an individual will depend upon the amount oftoxin the individual actually received from the bite. However, effectiveamounts for detoxifying or reducing the toxicity of snake neurotoxins inmice usually range from about 0.01 μg to 0.3 μg, preferrably about 0.02μg to 0.05 μg, of a reduced thioredoxin; from about 0.1 nanomole to 3.0nanomoles, preferably from 0.2 nanomole to 1.0 nanomole, of reducedlipoic acid in the presence of about 0.05 μg of a thioredoxin; fromabout 1.0 nanomole to 30 nanomoles, preferably from 2.0 nanomoles to 5.0nanomoles, of DTT, preferrably in the presence of 0.05 μg of athioredoxin, for every gm of mouse body weight.

The effective amounts for detoxifying a snake neurotoxin in a mouseusing the components of the NADP-thioredoxin system range from about0.01 μg to 0.3 μg, preferrably about 0.02 μg to 0.05 μg of athioredoxin; from about 0.005 μg to 0.12 μg, preferably from 0.01 μg to0.025 μg, of NTR and from about 5 nanomoles to 30 nanomoles, preferrably10 nanomoles to 15 nanomoles, NADPH for every gm of mouse body weight.

The preferred method of administering the NTS to an individual is alsoby multiple subcutaneous injections. The preferred thiol redox agent forhuman use is human thioredoxin administered via the NADP-thioredoxinsystem or with lipoic acid or DTT.

A partial list of the venomous snakes which produce the neurotoxinswhich can be inactivated or detoxified by the methods of this inventionappears on pages 529–555 of Chippaur, J.-P. et al. (1991) Reptile Venomsand Toxins, A. T. Tu, ed., Marcel Dekker, Inc., which is hereinincorporated by reference.

Other features and advantages of the invention with respect toinactivating and detoxifying venome can be ascertained from thefollowing examples.

EXAMPLE 26 Reduction Studies of Bee, Scorpion and Snake Venoms andLabeling with mBBr

Reactions were carried out with 50 μg venom (final volume of 100 μl) in30 mM Tris-CHl buffer pH 7.9 containing the following proteaseinhibitors: phenylmethylsulfonyl fluoride (PMSF), leupeptin andpepstatin (final concentrations used in the assay respectively: 100 μM,1 μM and 1 μM). With NADPH as a reductant, the mixture also contained 4μg thioredoxin, 3.5 μg NTR (both from E. coli) and 12.5 mM NADPH. Whenthioredoxin (4 μg, E. coli or human) was reduced by DTT, NADPH and NTRwere omitted and DTT was added to 0.5 mM. Assays with GSH were performedsimilarly but at a final concentration of 5 mM and in the presence of1.5 μg glutathione reductase and 12.5 mM NADPH. The mixture wasincubated for 20 min at room temperature, mBBr was then added to 1.5 mMand the reaction was continued for 15 min at room temperature. Thereaction was stopped and excess mBBr derivitized by adding 10 μl of 100mM β-mercaptoethanol, 5 μl of 20% SDS and 10 μl of 50% glycerol. Sampleswere then analyzed by SDS-polyacrylamide gel electrophoresis aspreviously described.

The same experiment with the NADP-thioredoxin system was performedwithout adding protease inhibitors.

After 20 min incubation at room temperature with different reductantsand in the presence of protease inhibitors, the samples were derivatizedwith mBBr and separated by electrophoresis and fluorescence wasdetermined. It was shown that in all cases thioredoxin (E. coli orhuman) specifically reduced components of the venoms. The gel alsoshowed that thioredoxin reduces venom components in a similar way whenthe reaction was performed in the absence of protease inhibitors.

The reduction of bee, scorpion and snake venoms by the NADP-Thioredoxinsystem with and without protease inhibitors was also shown using theSDS-Polyacrylamide gel mBBr procedure. After 20 min incubation at roomtemperature with NTS in the presence or absence of any proteaseinhibitors, the samples were derivatized with mBBr, separated byelectrophoresis, and fluorescence was determined as previouslydescribed.

Materials

Venoms: Been venom from Apis mellifera, scorpion venom from Androctonusaustralis, and snake venom from Bungarus multicinctus were purchasedfrom Sigma chemical Co. (St. Louis, Mo.).

Protease Inhibitors: Phenylmethylsulfonyl fluoride (PMSF), Leupeptin andPepstatin were purchased from Sigma Chemical Co. (St. Louis, Mo.).

Venom Detoxification

Detoxification of bee, scorpion and snake venoms is determined bysubcutaneous injection into mice. Assays are done in triplicate. Priorto injection, the venom is diluted in phosphate-saline buffer (0.15 MNaCl in 10 mM Na₂HPO₄/NaH₂PO₄ pH 7.2) at concentrations ranging up totwice the LD₅₀ (per g mouse): bee venom from Apis mellifera, 2.8 μg;scorpion venom from Androctonus australis, 0.32 μg; and snake venom fromBungarus multicinctus, 0.16 μg. At 5, 10, 30, 60 minutes and 4, 12 and24 hr after injection, separate groups of challenged mice are injected(1) intravenously and (2) subcutaneously (multiple local injectionsaround the initial injection site). The thioredoxin is reduced with: (1)E. coli NADP-thioredoxin system, using 0.08 μg thioredoxin, 0.07 μg NTRand 25 nmoles NADPH; (2) Thioredoxin reduced by DTT or reduced lipoicacid (0.08 μg E. coli or human thioredoxin added to 1 nmoledithiothreitol or 1 nmole of reduced lipoic acid). Concentrations areper μg venom injected into the animal; all solutions are prepared inphosphate-saline buffer.

The effect of thioredoxin on detoxification is determined by (1)comparing the LD₅₀ with the control group without thioredoxin and (2)following the extent of the local reaction, as evidenced by necrosis,swelling and general discomfort to the animal.

Reduction Studies for Reducing Snake Neurotoxins—Materials and Methods

Toxins

Porcine pancreas phospholipase A₂, erabutoxin b and β-bungarotoxin werepurchased from Sigma Chemical Co. (St. Louis, Mo.). As the phospholipaseA₂ was provided in 3.2 M (NH₄)SO₄ solution pH 5.5, the protein wasdialysed in 30 mM Tris-HCl buffer, pH 7.9, using a centricon 3 KDacutoff membrane. α-Bungarotoxin and α-bungarotoxin¹²⁵I were a kind giftfrom Dr. Shalla Verrall.

Reagents and Fine Chemicals

DL-α-Lipoic acid, L-α-phosphatidylcholine from soybean, NADPH andβ-mercaptoethanol were purchased from Sigma Chemical Co. (St Louis, Mo.)and monobromobimane (mBBr, trade name thiolite) from Calbiochem (SanDiego, Calif.). Reagents for sodium dodecylsulfate (SDS)-polyacrylamidegel electrophoresis were purchased from Bio-Rad Laboratories (Richmond,Calif.).

Proteins and Enzymes

Thioredoxin and NTR were purified from E. coli as is described by Jiaoet al., (1992) Ag. Food Chem. (in press). Thioredoxin h was purifiedfrom wheat germ (Florencio, F. J. et al. (1988) Arch Biochem. Biophys.266:496–507) and thioredoxins f and m from spinach leaves (Florencio, F.J. et al., supra.). Human thioredoxin was a kind gift of Dr. EmanuelleWollman. NADP-malate dehydrogenase was purified from corn leaves(Jacquot, J.-P. et al. (1981) Plant Physiol. 68:300–304) and glutathionereductase from spinach leaves (Florencio, F. J. et al., supra.). E. coliglutaredoxin was a kind gift of Professor A. Holmgren.

SDS-Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was performed in 10–20% gradientgels of 1.5 mm thickness that were developed for 3 hr at a constantcurrent of 40 mA. Following electrophoresis, gels were soaked for 2 hrin 12% (w/v) trichloroacetic acid and then transferred to a solutioncontaining 40% methanol and 10% acetic acid for 12 hr to remove excessmBBr. The fluorescence of protein-bound mBBr was determined by placinggels on a light box fitted with an ultraviolet light source (365 nm).Gels were photographed with Polaroid positive/negative Landfilm, type55, through a yellow Wratten gelatin filter No. 8 (cutoff=460 nm)(exposure time 40 sec. at f4.5). Gels were stained for protein for 1 hrin solution of 0.125% (w/v) Coomassie blue R-250 in 10% acetic acid and40% methanol. Gels were destained in this same solution from whichCoomassie blue was omitted.

Polaroid negatives of fluorescent gels and dry stained gels were scannedwith a laser densitometer (Pharmacia-LKB Ultroscan XL). The bands werequantified by evaluating areas or height of the peaks with Gelscan XLsoftware.

EXAMPLE 27 Reduction of Toxins and Labeling with mBBr

Reactions were carried out with 10 μg of target toxin in a final volumeof 100 μl in 30 mM.Tris-HCl buffer, pH 7.9, with 0.8 μg thioredoxin, 0.7μg NTR (both from E. coli) and 2.5 mM NADPH. When thioredoxin wasreduced by DTT, NADPH and NTR were omitted and DTT was added to 0.5 mM.Assays with GSH were performed similarly, but at a final concentrationof 1 mM. For reduction by glutaredoxin, the thioredoxin and NTR werereplaced by 1 μg E. coli glutaredoxin, 0.38 μg glutathione reductase(partially purified from spinach leaves), 1 mM GSH and 2.5 mM NADPH (thecombination of these four components is called NADP/glutaredoxinsystem). Reduction by the reduced form of lipoic acid, was carried outin a volume of 100 μl at two concentrations, 100 μM and 200 μM, bothalone and with 0.8 μg of thioredoxin. The mixture was incubated for 2 hrat 37° C. in the case of erabutoxin b and α-bungarotoxin, 1 hr at roomtemperature for β-bungarotoxin and 20 min at room temperature forphospholipase A₂. After incubation, mBBr was added to 1.5 mM and thereaction continued for 15 min at room temperature. The reaction wasstopped and excess mBBr derivatized by adding 10 μl of 100 mMβ-mercaptoethanol, 5 μl of 20% SDS and 10 μl 50% glycerol. Samples werethen analyzed by SDS-polyacrylamide gel electrophoresis.

Total toxin reduction was accomplished by boiling samples for 3 min in 2mM DTT. After cooling, the samples were labeled with mBBr and treated asbefore, except that all samples were again boiled for 2 min prior toloading in the gel. Dithiothreitol (DTT) and the reduced forms ofthioredoxin and lipoic acid are dithiol reductants as opposed tomonothiol reductants like 2-mercaptoethanol and glutathione. DTT is asynthetically prepared chemical agent, whereas thioredoxin and lipoicacid occur within the cell. Erabutoxin b was significantly reduced bythe NTS, DTT and thioredoxin and reduced lipoic acid and thioredoxin.With erabutoxin b lipoic acid was shown to be more specific reductantthan dithiothreitol. Dithiothreitol reduced the toxin partly withoutthioredoxin whereas reduced lipoic acid did not (lane 8). The resultsalso showed that the NTS or DTT plus thioredoxin are specific reductantsfor α-bungarotoxin and β-bungarotoxin.

EXAMPLE 28 NADP-Malate Dehydrogenase Activation

The ability of snake toxins to activate chloroplast NADP-malatedehydrogenase was carried out by preincubating 5 μg toxin with alimiting thioredoxin concentration (to restrict activation of the enzymeby the thioredoxin): E. coli thioredoxin, 0.25 μg; human, 0.9 μg; wheat,1.15 μg; spinachfand m, 0.375 and 0.125 μg, respectively. Purified cornNADP-malate dehydrogenase, 1.4 μg, was added to a solution containing100 mM Tris-HCl, pH 7.9, thioredoxin as indicated, and 10 mM DTT (finalvolume 0.2 ml). After 25 min, 160 μl of the preincubation mixture wasinjected into a 1 cm cuvette of 1 ml capacity containing (in 0.79 ml)100 mM Tris HCl, pH 7.9, and 0.25 mM NADPH. The reaction was started bythe addition of 50 μl of 50 mM oxalacetic acid. NADPH oxidation wasfollowed by monitoring the change in absorbance at 340 nm with a Beckmanspectrophotometer fitted with a four-way channel changer. The results ofthis experiment showed that the reduction by different reducedthioredoxins of erabutoxin b significantly alters the toxin's biologicalability to activate NADP-malate dehydrogenase. The results demonstratethat, although there are differences in effectiveness, all thioredoxinstested function to some extent in limiting the effect of the toxin.

EXAMPLE 29 Proteolysis assay of Erabutoxin b

Erabutoxin b, 10 μg was incubated for 2 hr at 37° C. with 30 mM Tris-HClbuffer pH 7.9 (total volume, 100 el). As indicated, the buffer wassupplemented with 0.8 μg thioredoxin, 0.7 μg NTR and 2.5 mM NADPH. Whenthioredoxin was reduced by DTT the NTR and NADPH were omitted and DTTwas added to 0.5 mM. Following incubation, samples were digested with0.4 and 2 μg of trypsin for 10 min at 37° C. DTT, 4.8 μl of 50 mMsolution, 5 μl of 20% SDS and 10 μl of 50% glycerol were added, sampleswere boiled for 3 min, and then subjected to SDS-polyacrylamide gelelectrophoresis. Gels were stained with Coomassie blue and the proteinbands quantified by densitometric scanning as described above. Theresults of the assay are shown in Table XVI below. These results showthat reduction of a snake neurotoxin (erabutoxin b) renders the toxinmore susceptible to proteolysis. An extension of this conclusion wouldindicate that administration of reduced thioredoxin as a toxin antidoteshould help to destroy the toxin owing to the increase in proteolyticinactivation by proteases of the venom.

TABLE XVI Susceptibility of the Oxidized and Reduced Forms of Erabutoxinb to Trypsin % Erabutoxin b digested Treatment 0.4 μg trypsin 2 μgtrypsin Control 0.0 34.1 Reduced, NTS 21.1 57.8 Reduced, DTT 3.1 40.6Reduced, DTT + Trx 28.0 71.8Erabutoxin b, 10 μg was preincubated for 2 hours at 37° C. in 30 mMTris-HCl buffer, pH 7.9, as follows: control, no addition; reduced by E.coli NADP/thioredoxin system (NTS), thioredoxin, NTR and NADPH; reducedby DTT, DTT; and reduced by DTT plus thioredoxin, DTT supplemented withE. coli thioredoxin. After preincubation 0.4 μg and 2 μg of trypsin wereadded to the indicated which then were analyzed by SDS-polyacrylamidegel electrophoresis.

EXAMPLE 30 Phospholipase A, Assay

Activity of the oxidized and reduced forms of the phospholipase A₂component of β-bungarotoxin was determined spectrophotometricallyfollowing change in acidity as described by Lobo de Araujo et al. (1987)Toxicon 25:1181–1188. For reduction experiments, 10 μg toxin wasincubated in 30 mM Tris-HCl buffer, pH 7.9, containing 0.8 μgthioredoxin, 0.7 μg NTR and 7 mM NADPH (final volume, 35 μl). After 1 hrincubation at room temperature, 20 μl of the reaction mixture was addedto a 1 cm cuvette containing 1 ml of assay solution (adjusted to pH 7.6)that contained 10 mM CaCl₂, 100 mM NaCl, 4 mM sodium cholate, 175 μMsoybean phosphatidylcholine and 55 μM phenol red. The reaction wasfollowed by measuring the change in the absorbance at 558 nm in aBeckman Du model 2400 spectrophotometer. The results of this experimentdemonstrated that β-bungarotoxin loses most of its phospholipaseactivity when reduced by thioredoxin. The results are consistent withthe conclusion that the administration of reduced thioredoxin followinga snake bite would help detoxify the toxin by eliminating phospholipaseA₂ activity.

EXAMPLE 31 α-Bungarotoxin Binding to Acetylcholine Receptor

α-Bungarotoxin binding was assayed with cultured mouse cells by usingradiolabeled toxin (Gu, Y. et al. (1985) J. Neurosci. 5:1909–1916).Mouse cells, line C₂, were grown as described by Gu et al (Gu, Y. etal., supra.) and plated in 24-well plastic tissue culture plates(Falcon) at a density of about 3000 cells per well. Growth medium wasreplaced by fusion medium after 48 hr and again after 96 hr. Cultureswere used for assay after an additional 2 days growth.

α-Bungarotoxin binding was determined with cells subjected to threedifferent treatments: [A] 10 nM α-bungarotoxin¹²⁵I (262 Ci/mmole) waspreincubated 2 hr at 37° C. in 200 μl of phosphate-saline buffer (0.15MNaCl in 10 mM Na₂HPO₄/NaH₂PO₄ pH 7.2) with 4 μg thioredoxin, 3.5 μg NTR(both from E. coli) and 6.25 mM NADPH. In certain cases, the NTR andNADPH were replaced by 1.25 mM DTT. After 2 hr incubation, the mixturewas transferred to a well containing mouse cells, washed two times withphosphate-saline, and incubated for 2 hours at 37° C. [B] After washingthe cells two times with phosphate-saline buffer, 10 nMα-bungarotoxin¹²⁵I (in 200 μl of phosphate-saline) was added per well.Following a 2 hr incubation at 37° C., cells were washed again withphosphate-saline buffer to remove unbound toxin. As indicated, 200 μlsaline, supplemented with 0.68 mM CaCl₂, 0.49 mM MgCl₂, 4 μgthioredoxin, 3.5 μg NTR and 6.25 mM NADPH were added to the well. Theplate was incubated 2 hr at 37° C. NTR and NADPH were omitted fromtreatment with DTT which was added at 1.25 mM. [C] After washing cellstwice with phosphate-saline buffer, 200 μl of a solution containing 4 μgthioredoxin, 3.5 μg NTR and 6.25 mM NADPH, were added to each well. Insome cases, NTR and NADPH were replaced with 1.25 mM DTT. The plate wasincubated for 2 hr at 37° C. Cells were then washed twice withphosphate-saline buffer to remove the added reductant. Phosphatesalinebuffer, 200 μl, containing 0.68 mM CaCl₂ and 0.49 mM MgCl₂ and 10 nMα-bungarotoxin¹²⁵I was added to each well. Incubation was continued for2 hr at 37° C. The results of this assay are shown in Table XVII. Thisexperiment shows that when reduced by thioredoxin, β-bungarotoxin can nolonger bind to the acetylcholine receptor. When extended to the wholeanimal, the thioredoxin-linked reduction mechanism would result indetoxification by eliminating binding of the toxin to its targetreceptor. Each α-bungarotoxin binding assay was done in triplicate.Nonspecific binding was measured by adding 100-fold excess unlabeledα-bungarotoxin to the incubation mixture. After the incubation period,the cells in all cases were washed with phosphate-saline to removeunbound toxin. The amount of toxin bound was determined by solubilizingthe cells in 0.1 M NaOH and measuring radioactivity in a gamma counter.

TABLE XVII Binding of α-Bungarotoxin to the Acetylcholine Receptor ofMouse Cells % Binding Treatment A

2 hr, 37° C. 2 hr, 37° C. No reductant 100.0 NTS 0.0 DTT plusThioredoxin 0.0 NTS minus NTR 63.0 NTS minus Thioredoxin 78.0 NTS minusNADPH 101.0 Treatment B

2 hr, 37° C. 2 hr, 37° C. No reductant 100.0 NTS 78.0 DTT plusThioredoxin 76.0 Treatment C

2 hr, 37° C. 2 hr, 37° C. No reductant 100.0 NTS 68.7 DTT 85.0 DTT plusThioredoxin 68.8 E. coli NTS: thioredoxin, NTR and NADPH

EXAMPLE 32 Example for Detoxification in an Animal

Detoxification of snake neurotoxins is determined by subcutaneousinjection into mice. Assays are done in triplicate. Prior to injection,the toxin is diluted in phosphate-saline buffer (0.15 M NaCl in 10 mMNa₂HPO₄/NaH₂PO₄ pH 7.2) at concentrations ranging up to twice the LD₅₀dose. (LD₅₀ is defined as that dose of toxin that kills 50% of a givengroup of animals.) For toxicity.tests, the following neurotoxinconcentrations correspond to the LD₅₀ (per g mouse): erabutoxin b, 0.05μg-0.15 μg; α-bungarotoxin, 0.3 μg; and β-bungarotoxin, 0.089 μg. At 5,10, 30, 60 minutes and 4, 12 and 24 hr after injection, separate groupsof the challenged mice are injected (1) intravenously, and (2)subcutaneously (multiple local injections around the initial injectionsite). The thioredoxin is reduced with: (1) the E. coli NADP-thioredoxinsystem, using 0.08 μg thioredoxin, 0.07 μg NTR and 25 nanomoles NADPH;(2) Thioredoxin plus 1–2 nanomoles of reduced lipoic acid, using 0.08 μgE. coli or 0.20 μg human thioredoxin, and (3) using 0.08 μg E. coli or0.20 μg human thioredoxin with 5 nanomoles dithiothreitol(concentrations are per μg toxin injected into the animal; all solutionsare prepared in phosphate-saline buffer).

The effect of thioredoxin on detoxification is determined by (1)comparing the LD₅₀ with the control group without thioredoxin; (2)following the extent of the local reaction, as evidenced by necrosis,swelling and general discomfort to the animal; (3) following the serumlevels of creatin kinase, an indicator of tissue damage. Creatin kinase,which is released into the blood as a result of breakage of musclecells, is monitored using the standard assay kit obtained from SigmaChemical Co. (St. Louis, Mo.).

The symptoms of snake bite are multiple and depend on a variety offactors. As a consequence, they vary from patient to patient. There are,nonetheless, common symptoms that thioredoxin treatment should alleviatein humans. Specifically, the thioredoxin treatment should alleviatesymptoms associated with neurotoxic and related effects resulting fromsnake bite. Included are a decrease in swelling and edema, pain andblistering surrounding the bite; restoration of normal pulse rate;restriction of necrosis in the bite area; minimization of the affectedpart. A minimization of these symptoms should in turn result inimprovement in the general health and state of the patient.

Reduction of Food and Pollen Proteins and Allergens

The invention provides a method for chemically reducing the disulfidebonds in major allergen proteins particularly food and pollen allergenproteins and for decreasing or eliminating the allergenicity that occurswhen foods containing those proteins are ingested or pollens containingthose proteins are inhaled, ingested or come in contact with mucusmembranes.

One method of decreasing or eliminating an allergic reaction involvessubjecting an animal over a period of time to immunotherapy with varyingdoses of an allergen that has been reduced by reduced thioredoxin. Theallergen may be a pollen protein or an allergen protein found in plantssuch as poison oak or in animals such as dust mites.

The invention also provides a method for increasing the digestibility ofallergen proteins and therefore food but also other proteins such aspollens that may be swallowed as well as inhaled. The disulfide bonds ofthe proteins were reduced to the sulfhydryl (SH) group by thioredoxin.The other major cellular thiol reductant, glutathione, was ineffectivein this capacity. The proteins are allergenically active in the oxidized(S—S) state; when treated with reduced thioredoxin they are reduced (SHstate) and lose allergenicity. Thioredoxin achieves this reduction whenactivated (reduced) either by NADPH via the enzyme NADP-thioredoxinreductase (physiological reducing system), or by dithiothreitol (DTT), asynthetic chemical reductant or by lipoic acid a physiologicalreductant. While use of NADPH as a reductant is often preferred inalmost all instances, physiologically acceptable lipoic acid may be usedand DTT may also be acceptable for the treatment of some proteins. Ifthe allergen contains thioredoxin then NADPH/NTR or lipoic acid may beused in some cases without added thioredoxin.

Presumably the proficiency of reduced thioredoxin to decrease oreliminate the allergenicity caused by the food depends upon the abilityof the reduced thioredoxin to reduce the intramolecular disulfide bondsin the allergenic proteins in the food. Also, an allergen that has hadits disulfide bonds reduced by thioredoxin will cause less allergenicity(i.e., it will be hypo-allergenic) but will still retain its ability tobe an effective immunotherapeutic agent.

Food proteins that have intramolecular disulfide bonds can be reducedand the allergenicity of foods containing these proteins can be at leastdecreased and the digestibility increased by reduced thioredoxin whenthe food is treated with the thioredoxin for an effective period of timeat an effective temperature. The effective temperature for incubatingthe food is from about 4° C. to 55° C. but any temperature which willallow the reduction of the disulfide bonds is acceptable. The effectivetime for incubation is from about 20 min to 2 hrs but again any timethat allows for reduction of the disculfide bonds without significantlydegrading food quality is acceptable. For example, the proteins may alsobe reduced and remain reduced by incubation at about 4° C. for about 48hrs.

Examples of the foods that will exhibit reduced allergenicity andincreased digestibility upon treatment with reduced thioredoxin arebeef, milk, soy, egg, rice, wheat and nuts.

A preferred method for decreasing the allergenicity and increasing thedigestibility of food ingested by a mammal is to pretreat or incubatethe food using the components in the NADP-thioredoxin system (NTS). Ingeneral, the effective amounts of the components range from about 0.01mg to 4.0 mg, preferably about 0.10 mg to 2 mg of thioredoxin; fromabout 0.01 mg to 4 mg, preferably from about 0.20 mg to 2 mg of NTR andfrom about 1 micromole to 250 micromoles, preferably about 25 micromolesto 100 micromoles of NADPH for every 1.0 gm of protein in the food. Ofcourse, the effective amounts of each component will vary depending uponthe amount of the other components, the time and temperature ofincubation, as well as the particular food and the amount of innatethioredoxin and its reductants in that food. For example, the aboveamounts of thioredoxin were determined on the basis of using 1 mg NTRand 100 micromoles NADPH per gram of food protein. The range of NADPHwas determined on the basis of using 1 mg NTR and 1 mg thioredoxin. Theappropriate component amounts may also be affected by prior treatment ofthe food and the type of animal and its condition.

The NADP/thioredoxin system is also able to reduce the disulfideallergens of airborne allergens such as ragweed pollen and grasses anddermal contact allergens such as dust mites and thereby alleviate theIgE response of an animal to those allergens when the animal receivesthese reduced allergens systemically.

The effective temperature for treating an allergen such as a pollenallergen with the NADP/thioredoxin system is from about 4° C. to 55° C.but again any temperature which will allow the reduction of thedisulfide bonds is acceptable. The effective time for incubation issimilar to the time for treating food (from about 20 min to about 1 hr)but again any time that allows for reduction of the disulfide bondswithout significantly degrading the protein is acceptable. For example,the proteins may also be reduced and remain reduced by incubation atabout 4° C. for about 24 hrs.

Examples of allergens that will exhibit reduced allergenicity andincreased digestibility upon treatment with reduced thioredoxin includedisulfide containing allergenic proteins such as ragweed pollens, grasspollens, poison oak and ivy allergens and dust mite allergen protein.

A preferred method for decreasing the allergenicity and alleviating theIgE response of a mammal to an allergen is to pretreat or incubate theallergen using the components in the NADP-thioredoxin system (NTS). Ingeneral, the effective amounts of the components for airborne andcontact allergens like food range from about 0.1 mg to 6.0 mg,preferably about 0.1 mg to 4.0 mg of thioredoxin; from about 0.1 mg to8.0 mg, preferably from about 0.2 mg to 7.0 mg of NTR and from about 1micromole to 500 micromoles, preferably about 25 micromoles to 400micromoles of NADPH for every 1 gm of protein of the allergen. Again,the effective amounts of each component will vary depending upon theamount of the other components, the time and temperature of incubation,as well as the particular allergen protein or allergen extract and theamount of innate thioredoxin and its reductants in that allergenextract. The above amounts of thioredoxin were determined on the basisof using 1 mg NTR and 100 micromoles NADPH per gram of allergen protein.The range of NADPH was determined on the basis of using 1.0 μg NTR and1.0 μg thioredoxin per 1.0 gm pollen protein.

Varying doses of the thioredoxin reduced allergen extract are theninjected into the animal over a prescribed period of time.

The appropriate component amounts may also be affected by priortreatment of the allergen, the particular allergen and the type ofanimal and its condition.

Other features and advantages of the invention with respect todecreasing the allergenicity and increasing the digestibility of aparticular allergen can be ascertained from the following examples.

EXAMPLE 33 Treatment of Milk, Soy, Wheat and Beef with Thioredoxin

For this study a 1 to 5 physiological saline (PBS) dilution of a stock,1:20 weight/volume (w/v), cow's milk allergenic extract (Catalogue No.3390JG, Miles, Inc., Elkhart, Ind.), a 1 to 10 PBS dilution of a stock,1:10 w/v, soy allergenic extract (Catalogue No. 3597ED, Miles, Inc.,Elkhart, Ind.) and a 1 to 5 PBS dilution of a stock, 1:10 w/v, wheatallergenic extract (Catalogue No. 3708ED, Miles, Inc., Elkhart, Ind.)were prepared. A 1 to 5 PBS dilution of a stock, 1:10 w/v, commercialbeef allergenic extract (Catalogue No. 3078JF, Miles, Inc., Elkhart,Ind.) was also prepared.

In the case of cow's milk, 0.1 ml of the dilution was treated with theNADP/thioredoxin system (NTS) which comprised incubating the allergen inthis instance with 4.8 micrograms thioredoxin, 4.2 micrograms NTR and 1mM NADPH (final volume, 0.2 ml). A second sample was also prepared using0.1 ml of the diluted milk allergen incubated with 1.5 mM DTT and 4.8micrograms thioredoxin (final volume, 0.2). In all the allergen studies,including the studies in this Example and the ones following, thethioredoxin and NTR used were purified as previously described from E.coli that had been transformed to overproduce those proteins (de laMotte-Guery, F. et al. (1991) Eur. J. Biochem. 196:287–294, and Russel,M. et al. (1988) J. Biol. Chem. 263:9015–9019). For soy and wheat, 0.05ml of the dilutions were incubated with 2.4 micrograms thioredoxin, 2:1micrograms NTR and 1 mM NADPH. In addition, an identical treated controlsample of PBS was prepared. DTT treated soy and wheat samples were alsoprepared using 0.05 ml of the separate allergen dilutions incubated with1.5 mM DTT and 2.4 micrograms thioredoxin. The final volume for thecontrol and all the soy and wheat samples was 0.1 ml. The milk and wheatpreparations were incubated at room temperature for 25 min while the soypreparation was incubated at 37° C. for 1 hr and 25 min. With the beef,0.05 ml of the dilution was incubated with 2.4 micrograms thioredoxin,2.1 micrograms NTR and 1 mM NADPH (final volume, 0.1 ml) for 25 min at37° C. Another 0.05 ml sample was treated the same way but at roomtemperature. Following incubation 1 ml dilutions ranging from 1×10³ to1×10⁶ or from 1×10³ to 1×10⁷ were prepared for each treated food extractpreparation. The diluted samples were used for testing within 30 min.

EXAMPLE 34 Determination of the Reduction of Food Allergens by theNADP/Thioredoxin System and Increase in Proteolysis Using the mBBrFluorescent Labeling/SDS-Polyacrylamide Gel Electrophoresis

Method

For this study a 1 to 2.5, 1 to 5 and 1 to 1.5 dilution with PBS of thestock cow's milk, beef and wheat extracts described in Example 33 wererespectively prepared. To 50 microliters of these dilutions were added2.4 micrograms thioredoxin, 2.1 micrograms NTR and 1 mM NADPH (finalvolume, 0.1 ml). The controls consisted of 50 microliters of theparticular diluted extract and 50 microliters of PBS. The preparationswere incubated for 25 min at room temperature and also at 37° C. Afterincubation, 8 μl of 80 mM mBBr was added and the reaction continued for15 min at room temperature. The reaction was stopped and excess mBBrderivatized by adding 10 μl of 100 mM β-mercaptoethanol, 10 μl of 20%SDS and 10 μl of 50% glycerol. The samples were analyzed by themBBr/SDS-polyacrylamide gel electrophoresis technique previouslydescribed. The results showed that the NTS effectively reduced theproteins in the allergenic extracts at both room temperature and 37° C.In an additional study, where a PBS dilution of the soy stock extractdescribed in Example 33 was similarly treated with the NTS, an analysisusing the mBBr labeling/SDS-PAGE method showed that thioredoxin alsoreduced the soy proteins. However, when soy, cow's milk, wheat, egg andbeef allergenic proteins were incubated with glutathione, glutathionereductase and NADPH, there was minimal or no reduction of those treatedallergenic proteins.

A PBS dilution of a commercial rice allergenic extract (Catalogue No.3549ED, Miles, Inc., Elkhart, Ind.) is also similarly incubated with theNTS and analyzed using the mBBr/SDS-PAGE technique to show that reducedthioredoxin reduces rice allergen proteins.

In a separate study, it was also observed that food allergen proteinsfrom the commercial extracts described in Example 33 that had beenreduced by the NTS and were further incubated with trypsin had anincreased susceptibility to proteolysis over controls that had not beentreated with the NTS. The analysis of the reduction was done using themBBr/SDS-PAGE techniques. Further in this study, when 10 μg of an NTSreduced purified milk allergen protein, β-lactoglobulin (Sigma ChemicalCo.), was treated with 2 μg of trypsin, proteolysis was 100% as comparedwith only 50% for the identically trypsin treated oxidizedβ-lactoglobulin. When 10 μg of another purified milk allergen, oxidizedα-lactalbumin (Sigma Chemical Co.), was similarly treated with 2 μgtrypsin, there was no noticeable proteolysis. However, 10 μg of purifiedα-lactalbumin reduced by the NTS was proteolyzed 80% by trypsin. Also 10μg of α-lactalbumin reduced by 0.8 μg of thioredoxin and 0.5 mM DTT was100% proteolyzed by 2 μg of trypsin.

EXAMPLE 35 Reduction of Egg White Proteins

Dried chicken egg white was purchased from Sigma Chemical Co. About 80%of the total proteins in egg white are allergens. A solution of 20 mg/mlegg white was resuspended in PBS. Since not all the material wasdissolved, it was centrifuged at 14,000 RPM for 2 min. The soluble eggwhite proteins were used for the reduction study using mBBr fluorescentlabelling and SDS-polyacrylamide gel electrophoresis analysis. Thetreatments used were the control (no reductant), NTS, DTT plusthioredoxin, reduced glutathione (GSH) and reducedglutathione/glutathione reductase/NADPH. Reactions were carried out inPBS with 23 microliters of the soluble proteins from the 20 mg/ml eggwhite suspension in a final volume of 100 microliters. In the NTS, 7.5mM NADPH, 2.4 micrograms thioredoxin and 2.1 micrograms NTR were used.When thioredoxin was reduced by DTT, NADPH and NTR were omitted and DTTwas added to 1.5 mM. The GSH concentration used was 3 mM. For reductionby the GSH/GR/NADPH system, 3 mM GSH, 4 micrograms GR and 7.5 mM NADPfiwere used. The mixture was incubated for 1 hr at room temperature. Afterincubation, 7 microliters of 80 mM mBBr was added and the reactioncontinued for 15 min at room temperature. The reaction was stopped andexcess mBBr was derivatized by adding 10 microliters of 100 mMmercaptoethanol, 10 microliters of 20% SDS and 10 microliters of 50%glycerol. Samples were then analyzed by SDS-polyacrylamide gelelectrophoresis. The results of this experiment showed that the NTS andDTT plus thioredoxin are very effective in reducing egg white proteinswhich are about 80% allergens. GSH or GSH/GR/NADPH showed the same levelof reduction as the control and therefore is an ineffective reductant ofegg white proteins.

EXAMPLE 36 Sensitization of Animals for Allergenicity Studies

The animals used in this study were atopic dogs born to different pairsof littermates from an in-bred colony of high IgE-producing spaniels.

A litter of 9 pups (4 males, 5 females) was born to an in-bredIgE-responder bitch sired by her brother. On newborn day 1, for thecow's milk, soy and rice studies, a nine-pup litter was divided into twogroups: Group I of 5 pups was injected subcutaneously (SQ) in the rightaxilla with 1 μg the commercial soybean extract described in Example 33in 0.2 ml alum; Group II of 4 pups was injected SQ in the right axillawith 1 μg of commercial dried cow's milk extract (described in Example33) solubilized in 0.2 ml saline and 0.2 ml alum. All 9 pups were alsogiven 1 μg of a stock 1:10 w/v rice allergenic extract (Catalogue No.3549ED, Miles, Inc. Elkhart, Ind.) in 0.2 ml alum SQ in the left axilla.

At ages 3, 7 and 11 weeks, all pups were vaccinated with 0.5 ml of liveattenuated distemper-hepatitis vaccine (Pitman-Moore, Washington'sCrossing, Pa.) in the shoulder SQ. Two and 9 days after eachvaccination, they were given the same food antigens that they receivedin the neonatal period, e.g., 5 dogs in Group I received 1 μg soybeanextract in 0.2 ml alum, 4 dogs in Group 11 received 1 μg cow's milk in0.2 ml alum, all 9 pups received 1 μg rice extract in 0.2 alum SQ in theright and left axilla, respectively.

A ¹²⁵I-labelled rabbit anti-canine IgE serum (Frick, O. L. et al. (1983)Am. J. Vet. Res. 44:440–445) was used in a RAST assay. Cyanogen-bromideactivated filter paper discs were reacted with 100 μg of either soy,cow's milk or rice antigen, as in standard RAST protocol (Wide, L. etal. (1967) Lancet 2:1105–1107). A pool of newborn canine cord sera fromnon-atopic mongrel pups was used as a negative control or baseline cpm.

The pups were nursed for 6 weeks and weaned onto regular Puppy Chow(Ralston-Purina Company, St. Louis, Mo.) which included the sensitizingproteins, soybean meal, dried whey, and rice hulls; they were fedonce/day and given water ad lib, under veterinary care and supervisionat the University of California, Davis, Animal Resources Services,School of Veterinary Medicine. After 6 months, they were fed regularField & Farm Dog Chow.

Between 3 and 4 months of age, when it was found that the pups weremaking IgE antibodies to a particular food antigen, all 9 pups in thelitter were given double-blinded, 240 ml of either soy or cow's milkinfant formula, tofu, rice gruel, or vanilla-flavored Vivonex proteinhydrolysate (Norwich-Eaton Co., Norwich, Conn.) in the early morning.Abdominal girth was measured at umbilical level before the challenge andat 2 hours intervals during the day. They were observed closely by aveterinary technician for clinical signs of itching or rash, vomitingand frequency and character of stools, and for cough or respiratorydistress and nasal discharge. They were monitored for such signs ofreaction for 4 days. The next challenge feeding was given 7 days later.

All 9 pups in the litter gained weight and developed normally with nomedical problems. They had no diarrhea or other gastrointestinal signsunless they were challenged with the food they had been sensitized to.Also no skin or respiratory abnormalities occurred. There were nountoward reactions to the vaccinations or immunizations.

Canine IgE-RASTs to the 3 food proteins were followed at fortnightlyintervals with venous blood sampling.

Significantly more IgE-anti-food antibodies were produced by thecorresponding milk (p<0.0005 when challenged at 4 months) and soy(p<0.0005 for the first 3 months) immunized animals than by controls.The average titer for IgE-anti-rice-antibodies rose at 5–10 week,plateaued and then rose again sharply after 20–30 weeks of age. Alsostatistically significant reactions of diarrhea and abdominal bloatoccurred in the soy and milk immunized animals when they wererespectively fed soy and milk in their chow.

Another group of 8 dogs allergic to cow's milk, soy, wheat and beef werealso developed in a manner similar to the method described above. Fourlittermate pups from another litter in the described in-bred colony ofhigh IgE-producing spaniels were injected SQ in the right axilla with 1μg each of the stock cow's milk, soy, wheat and beef allergenic extractsdescribed in Example 33. Four pups from the same litter acted ascontrols. All eight pups were also given 1 μg of the rice extract in 0.2ml alum SQ in left axilla. The pups were vaccinated as above and at twoand nine days after each vaccination, were given the same food antigensin the same amount that they received as neonates. As above, they werefed foods which contained the appropriate sensitizing proteins (i.e.,cow's milk, soy, beef and wheat) in a similar schedule. As with theprevious soy and milk allergic animals, these immunized dogs producedsignificantly more anti-specific food IgE antibodies (including wheatand beef antibodies) than the controls. Again statistically significantreactions of diarrhea and abdominal bloat occurred in the immunizedanimals when they were challenged with food containing wheat, beef, soyor cow's milk.

EXAMPLE 37 Skin Test Determinations of the Decrease of Allergenicity inFood Allergens Treated with Reduced Thioredoxin

Aliquots of 100 microliters of each dilution of the thioredoxin treatedfood allergen dilutions described in Example 33 were injectedintradermally on the abdominal skin of the appropriate sensitized dogsdescribed in Example 36 (e.g., cow's milk sensitized dogs were injectedwith the cow's milk dilutions). Prior to the allergen dilutioninjections, the dogs' forelimb veins were injected with 4 ml of 0.5%Evans blue dye. Dogs exhibiting an allergenic reaction developed bluecolored wheals in the area of the allergen injection. After 10 minutesof development the size of the wheal (length and width) were measured.The observed size of the wheal and the dilution end point followinginjection of a concentration range of each allergen preparation wereused as the allergenicity indicator. It was found that thioredoxintreatment gave 50% protection with 1×10⁵ dilution of soy, fullprotection with a 3×10⁴ dilution of milk, full protection with a 1×10⁶dilution of wheat and at least partial protection with a 1×10⁵ dilutionof beef (see FIGS. 1, 2, 3, 4 and 5 respectively).

EXAMPLE 38 Feeding Test Determinations of the Allergenicity of FoodAllergens Treated with Reduced Thioredoxin

Approximately one week prior to feeding, dogs sensitized in the mannerdescribed in Example 36 were skin tested intradermally as in Example 36with the appropriate food allergen using the commercial allergenicextracts described in the previous examples. Based on these results,animals were separated into “control” and “thioredoxin-treated” groups.Each group was made up of representatives with complementarysensitivities—i.e., an equal number of strong, medium and weak reactorswas selected for each group. Unless indicated otherwise, the groups werecomprised of 3 animals (6 animals per experiment). For 3 days before and5 days after the feeding challenge, dogs were maintained on a Hill'sPrescription Diet Canine d/d diet (Hill's Division of Colgate PalmoliveCo., Topeka, Kans.). Dogs were observed throughout this period forclinical symptoms such as retching and vomiting. In addition, stoolswere monitored and scored as an indicator of the allergenic response tothe food being tested. The dogs' stools were counted for 3 days beforeand 3 days after the allergenic diet challenge and their consistency wasindicated by a number: 1=firm, 2=soft, and 3=runny. The stool score wasthen calculated by multiplying the number of stools time the consistencyfactor. As an indicator of the allergenic response to the food allergensbeing tested, the final net average stool score per day for each group(“control”, or “thioredoxin treated”) was calculated by subtracting theaverage stool score per day before from that after the allergenic dietchallenge. A higher net average stool score per day represents astronger allergenic response.

The procedures used for preparing and administering the diets is givenbelow. Unless indicated otherwise, reactions were carried out at roomtemperature.

Soy

Commercial soy formula (Isomil supplemented with iron, RossLaboratories, Columbus, Ohio), 1.026 kg, was dissolved in 3 l of water.The solution was separated into two lots of 1.925 l, one used as thecontrol and the other treated with the NADP/thioredoxin system (NTS) asfollows. A mixture containing 45 micromoles NADPH, 564 microgramsNADP-thioredoxin reductase (NTR) and 1.125 mg thioredoxin (all dissolvedin 30 mM Tris-HCl buffer, pH 7.9) was preincubated for 5 min and thenadded to one of the 1.925 l formula lots (henceforth the“thioredoxin-treated” lot). After adding the thioredoxin system,incubation was continued for an additional hour with constant stirring.An equal amount of buffer was added to the control lot. Afterincubation, 600 ml of formula was fed to each of the 3 dogs of theassigned group. The portions fed to the animals were equivalent to 25.0gm of soy protein prior to incubation.

Wheat

Unbleached flour, 1.5 kg, was added to 3 l of water, previously heatedto 37° C., in a gallon-size Waring blender. After 1 min blending, theflour suspension was divided equally into two lots, one was used as thecontrol and the other treated with the NADP/thioredoxin system asfollows. A mixture containing 45 micromoles NADPH, 564 micrograms (NTR)and 1.125 mg thioredoxin, all dissolved in 30 mM Tris-HCl buffer, pH 7.9was preincubated for 5 min and then added to one lot (henceforth the“thioredoxin-treated” lot). An equivalent amount of buffer was added tothe control lot. Both preparations were incubated 1 hr at 37° C. withfrequent stirring. A volume of 600 ml was removed from each preparation,mixed with one can (15 ¾ oz) d/d, wheat free, rice/lamb based, dog foodand fed to the 3 dogs of the assigned lot. Again these portions wereequivalent to 25 gm of wheat protein. In the experiment with 8 dogs, theflour was increased to 2.0 kg and the procedure scaled up accordingly.

Milk

Preparations of dried CARNATION that had been reconstituted with waterwere similarly incubated with the NADP/thioredoxin system. As before 3of the dogs received untreated and 3 received the treated milk. Thefinal portions that the dogs received were equivalent to 10 gm of milkprotein.

Results

The levels of components of the NADP/thioredoxin systems that were usedwere significantly higher than in the dough studies described above inExample 15. In this feeding study, preliminary trials indicated thathigher levels of these compounds were required to reduce the allergenicproteins as determined in vitro by the mBBr/SDS-polyacrylamide gelprocedure. The amounts of each component of the NADP/thioredoxin systemused for each dog per gm protein in the feeding trials is indicatedbelow relative to the amounts used in the baking tests:

Wheat Flour, Milk, Soy Formula* Thioredoxin 5-X NTR 5-X NADPH 2-X*Amount indicated are relative to those used in the above describedbaking tests in which 3 micrograms NTR and 0.3 micromoles NADPH wereadded per gram of flour protein. In the banking tests, loaves were bakedwith approximately 200 g flour of approximately 20 gm of flour protein.For the feeding experiments, food preperations were incubated withcomponents of the NADP/thioredoxin system for one hour either at roomtemperature (milk and soy) or 37° C. (wheat).

Based on “bedside symptoms” (vomiting and retching) as well as “stoolscore” (see FIG. 6), thioredoxin treatment was found to decrease theallergenic response of the dogs to the soy and wheat formulas. Theallergenicity of the milk formula may also be decreased by treatmentwith the NTS. It should be noted that while the thioredoxin and NTR usedwere from E. coli, thioredoxins from other sources such as yeast andthioredoxin h and m may also be used.

EXAMPLE 39 Determination of the Increased Digestibility and DecreasedAllergenicity of Thioredoxin Treated Milk Proteins and Raw Cow's Milk

Milk allergy is caused by several proteins—α-lactalbumin, serum albumin,caseins, and particularly β-lactoglobulin (BLG).

This example showed that the disulfide bonds of milk proteins andallergens were selectively and specifically reduced by thioredoxin. Oncereduced, the most active of these allergens (BLG) showed not only adecrease in allergenicity, but also a striking increase indigestibility. The susceptibility of other milk disulfide proteins topepsin also increased to some extent following reduction by thioredoxin.

Materials and Methods

Allergen Source

Raw cows milk was obtained from the experimental farm, University ofCalifornia at Davis. Pure β-Lactoglobulin A and B and an 80% mixture ofthe two forms were purchased from Sigma Chemical Co., St. Louis, Mo.

Animals

Dogs obtained from the same colony of inbred, high IgE-producing atopicdogs used in Examples 36–38 were sensitized and maintained at the AnimalResources Service, School of Veterinary Medicine, University ofCalifornia, Davis. These dogs, sensitized at birth, have been selectedfor a genetic predisposition to allergy and have a 15-year history offood and pollen hypersensitivity.

Chemicals and Enzymes

Reagents for sodium dodecyl sulfate/polyacrylamide (SDS/PAGE) wereobtained from Sigma, Boehringer Mannheim, Indianapolis, Ind. and BioradLaboratories, Hercules, Calif. DTT was purchased from BoehringerMannheim and monobromobimane (mBBr), from Calbiochem, San Diego, Calif.Thioredoxin and NTR were purified from E.coli strains overexpressing theproteins. Glutathione reductase was purified from spinach leaves by thesame procedure used for spinach NTR. Pepsin from porcine stomach mucosa,NADPH and other biochemical reagents were purchased from Sigma.

Food Sensitization of Atopic Dogs

Newborn pups from two litters of the atopic dog colony, designated CGBand GCB, were injected subcutaneously at day 1 with 1 μg each of wheat,cows milk and beef extract (Miles, Inc., Elkhart, Ind., described inExample 33) in 0.2 ml of alum. A third litter, designated CBB, wasinjected with a soy extract (Miles, Inc. described in Example 33) inaddition to these same allergens. Procedures for the sensitization,testing and maintenance of the pups were substantially the same as thosedescribed in Example 36.

Skin Tests

About 3 min prior to skin testing, each dog received 4–5 ml of filtered0.5% Evans blue dye solution (equivalent to 0.2 ml of 0.5% Evans bluedye per kg of weight) through a cephalic vein to enhance assessment ofthe cutaneous IgE antibodies. Serial dilutions of 100 μl of each samplewere injected intradermally on the abdominal skin to establish thetiter. After 15–20 min, the allergic response was determined bymeasuring the size of the blue wheal reaction (maximum length andwidth). An appropriate negative control (buffer diluted in physiologicalsaline) was included for each animal tested. Repeated tests withthioredoxin, NTR, NADPH and pepsin alone were consistently negative.

Protein Assay

Protein concentration was determined by the Bradford method using bovinegamma globulin as the standard. Concentration of pure BLG was determinedby its absorbance at 278 nm using a calculated molar extinctioncoefficient of 16800 (Gill, S. C. et al. (1989), Anal. Biochem.182:319–326).

Protein Modeling

A model of BLG structure, determined by Brownlow et al. at 1.8 Åresolution, was provided by the Protein Data Bank, Brookhaven NationalLaboratory (Brownlow, S. et al. (1997), “Bovine beta-Lactoglobulin at1.8 Resolution-Still an Enigmatic Lipocalcin”, Structure 5:481–495). Amodel of the protein with single mutated C160S (partly reduced) anddouble mutated C160S-C106S (fully reduced), was built by the Swiss-Modelprogram (Peitsch, M. C. (1996), “ProMod and Swiss-Model: Internet-basedtools for automated comparative protein modelling”, Biochem. Soc. Trans.24:274–279). A three dimension model of BLG was visualized by the RasMolprogram v2.6.

Protein Reduction

Reduction of the protein disulfide bonds was performed, in a volume of100 μl with either: (i) the NADP/thioredoxin system (NTS), consisting of5 μl of 25 mM NADPH, 8 μl of 0.3 mg/ml E. coli thioredoxin (i.e., 2.4 μgtotal thioredoxin) and 7 μl of 0.3 mg/ml E. coli NTR; or (ii) theNADP/glutathione system (NGS), composed of 5 μl of 25 mM NADPH, 10 μl of30 mM reduced glutathione (GSH) and 15 μl of 0.1 mg/ml glutathionereductase. Reactions were carried out in physiological buffered salinesolution (PBS; i.e., 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 2.7 mM KCl and 137 mMNaCl, pH 7.4) containing either 10 μg of pure target protein or 50 μg ofraw milk. The reaction mixtures were incubated at 4° C. overnight or at37° C. and 55° C. for 45 min. For complete reduction, samples wereincubated in PBS containing 5 μl of 100 mM DTT and boiled 5 min. Thereduced proteins were visualized on gels by mBBr labeling and gelelectrophoresis as described below. The extent of reduction wasdetermined by scanning the gels.

Pepsin Assay

BLG, 640 μg, or milk, 1 mg protein (i.e., about 30 el), was incubatedwith or without the thioredoxin system (NTS) at either 4° C. (to yieldfully reduced form) or 55° C. (to yield partly reduced form), under theconditions described above. The BLG, 320 μg, or milk, 500 μg protein,was then treated in 200 μl of simulated gastric fluid (SGF) as describedby Astwood, J. D. et al. (1996), “Stability of food allergens todigestion in vitro”, Nature Biotechnol. 14:1269–1273. SGF consists of0.32% pepsin (w/v) and 30 mM NaCl adjusted at pH 1.2 with HCl (Board ofTrustees (ed.) 1995, Simulated Gastric Fluid, TS., pp. 2053 in theUnited States Pharmacopeia 23, The National Formulary 18. United StatesPharmacopeial Convention, Inc. Rockville, Md.). The reaction mixture wasincubated at 37° C. and stopped by adding 0.375-fold volume of 160 mMNa₂CO₃ (ca. pH 7) after 0, 0.25, 1, 2, 4, 8, 15, 30 and 60 minincubation. The protein mixture was then subjected to SDS-PAGE (15%gels) and stained for protein with Coomassie blue as described below. Asindicated, the allergenicity of the digested samples was determined byskin test analysis.

Feeding Challenges

Reduction of 80% pure BLG (a mixture of the A and B forms) was carriedout for each dog in 100 ml of water. Each gram of BLG was treated byadding an aqueous mixture of 104 mg of NADPH, 1 mg of E. colithioredoxin and 1 mg of E. coli NTR. The reactions occurred in a shakerat 125 rpm at 37° C. for 45 min. Samples were stored overnight at 4° C.The following day untreated (control) or treated BLG, 2.5 gm was mixedwith a 12 oz. can of P/D food (Hills) and fed to a dog. Unchallengedanimals received dog food alone without BLG while control animalsreceived dog food with untreated BLG. Dogs were initially fed ¼ can ofuntreated food to initiate gastric flow. After 15 min, they were thenfed at 3 intervals separated by 15 min ⅓ of a can of food that had beenmixed with 2.5 g of either untreated or thioredoxin treated BLG. Duringthese intervals dogs were monitored and their GI response assessed. Dogswere then observed and their response recorded at hourly intervals forthe next 5 hr.

Data Analysis

The digestion response of the food challenged dogs described above wasassessed by assigning numbers to the timing, volume and fluidity ofvomit induced by feeding. Fluidity: no vomit=0, solid vomit=1, liquidvomit=2, liquid with blood or bile vomit=3. Volume: no vomit=0, smallvomit=1 and large vomit=2. Timing: delayed vomit=1, immediate vomit=2.

mBBr Labeling and Analysis of Proteins

Sulfhydryl groups were visualized as their fluorescent mBBr derivatives.mBBr, 10 μl of a 100 mM solution, was added to each protein sample.After 20 min of incubation, the reaction was stopped by adding 10 μl of100 mM 2-mercaptoethanol, 10 μl of 20% SDS and 20 μl of SDS/PAGE samplebuffer containing 80% (v/v) glycerol and 0.005% bromophenol blue.Proteins were then separated by SDS/PAGE (10–20% acrylamide gradient) asdescribed below. After electrophoresis, gels were placed in 12%trichloroacetic acid for 1 hr for fixation and then soaked overnight orlonger in 40% (v/v) methanol and 10% (v/v) acetic acid with severalchanges to remove excess mBBr. The destained gels were then placed under365 nm UV light to visualize fluorescent bands. Pictures were taken byeither (i) Polaroid photographs (Positive/Negative Landfilm, type 55)through a yellow Wratten gelatin filter no. 8 with an exposure time of45 s at f4.5, or (ii) the Nucleovision system of NucleoTech Corporation.

SDS/PAGE

Gels (10–20% acrylamide gradient, 1.5 mm thickness or 15% acrylamide,0.75 mm thickness) were prepared according to Laemmli, U. K. (1970),“Cleavage of structural proteins during the assembly of the head ofbacteriophage T4”, Nature 227.680–685. After electrophoresis, gels werestained with 0.01% Coomassie brilliant blue R-250 in 40% methanol and10% acetic acid for 1 hr and destained overnight with a solution of 20%ethanol and 10% acid acetic. Pictures were taken after destaining byeither (i) Polaroid photograph (exposure time 1/15 s at f7) or (ii) theNucleovision system of NucleoTech Corporation.

Sequence Analyses

The different forms of BLG (oxidized, partly and fully reduced, 10 μg)were separated as mBBr derivatives by SDS-PAGE (10–20% acrylamidegradient, 1.5 mm thickness or 15% acrylamide, 0.75 mm thickness). Anin-gel digestion method was used to obtain peptides containing cysresidues that would allow structural characterization (Hwang, B. J. etal. (1996), “Internal sequence analysis of proteins eluted frompolyacrylamide gels”, J. Chromatogr. B. Biomed. Appl. 686:165–175). Thedried gel used as starting material was placed in water to allowswelling and the cellophane layer was removed. The appropriate BLG bandswere then cut out from the reconstituted gel. Each protein band wasdiced into 1 to 2 mm pieces. In brief, gel pieces were dehydrated in aspeedvac, rehydrated in 0.1 M Tris buffer, pH 9 and 0.05% SDS, pH 9.0,containing 0.03 to 0.05 μg Lys-C endopeptidase (Wako) and incubatedovernight at 30° C. Peptides were eluted by extracting the gel twice for2 hr with water and twice for 2 hr with 70% acetonitrile/0.1%trifluoroacetic acid (TFA). The pooled extracts were dried, dissolved ina minimal volume of 6 M guanidine HCl-Tris, pH 8.2. Extracted peptideswere then reduced with dithiothreitol (DTT) and alkylated withiodoacetamide. After the reaction, the mixture was diluted 4-X withwater and the reduced guanidinium dodecylsulfate precipitate was removedby centrifugation. In-solution digestion was performed with trypsin inthe diluted reaction mixture for complete digestion. Peptides werepurified using a C18 microbore column (1 mm×15 cm, VYDAC) using anApplied Biosystems 172 HPLC system. After injection of sample, thecolumn was washed with 95% solvent A (0.1% TFA in water)/5% solvent B(70 acetonitrile/0/075% TFA) for 10 min using a flow rate of 80 μl/min.Peptides were eluted with a gradient of 5 to 70% Solvent B for 90 min.Purified peptides were sequenced using either an ABI 477 or 470Asequencer with on-line HPLC identification of thenylthiohydratoin (PTH)amino acids. Peptides containing either Cys 160 and Cys 119 wereisolated as indicated below. The disulfides of the fully oxidizedprotein correspond to Cys 106-Cys 119 and Cys 60-Cys 160. To identifythe disulfide bonds involved in the partly and fully reduced forms,trypsin peptide (s) unique to each were isolated. The partly and fullyreduced forms both showed the peptide:-Leu₁₄₉-Ser-Phe-Asn-Pro-Thr-Gln-Leu-Glu-Glu-Gln-Cys ₁₆₀His-Ile₁₆₂. Thefully reduced protein showed in addition the following peptide:-Tyr₁₀₂-Leu-Leu-Phe-Cys₁₀₆-Met-Glu-Asn-Ser-Ala-Glu-Pro-Glu-Gln-Ser-Leu-Ala-Cys₁₁₉-Gln-Cys₁₂₁-Leu-Val-.

Results

Reduction of Milk Proteins by the Thioredoxin System.

Thioredoxin was effective in the reduction of the most prevalentdisulfide protein of milk, namely BLG. To this end, the redox state ofpure BLG (A and B forms) was monitored following treatment with theNADP/thioredoxin system (NTS), consisting of NADPH, NTR and thioredoxin.As described above, samples were incubated with the NTS system and thenanalyzed using the monobromobimane (mBBr)/SDS-polyacrylamide gelelectrophoresis (PAGE) procedure. Here, as previously stated, thereduced (—SH) form of a target protein derivatized with mBBr andseparated by SDS-PAGE, appears as a fluorescent band when viewed inultraviolet light. It was found that, as seen in the previous Exampleswith a spectrum of proteins containing intramolecular disulfide bonds,the A and B forms of pure BLG were actively reduced by thioredoxin. Whenapplied to milk, thioredoxin not only reduced BLG but also several otherproteins, including α-lactalbumin as determined by the SDS/PAGE/mBBrlabeling procedure used for the gel in FIG. 7. For this gel 50 μg of rawmilk in PBS was applied to all lanes. The other variables for each laneare: Lane 1, control at 4° C., no addition; Lane 2, NTS, 55° C.; Lane 3,NTS, 4° C.; Lane 4, NGS, 55° C.; Lane 5, NGS, 4° C.; Lane 6, 5 mM DTT,100° C., 5 min; Lane 7 was the same as Lane 6 except it was stained withCoomassie blue. Note that the excess of NADPH, NTR and thioredoxinmaintained the target milk proteins in the reduced state throughout theexperiment. The minor band traveling in front of the major (24 kDa)casein components (α and β) is κ-casein.

Temperature was found to have an interesting and useful effect onreduction. When treated at 55° C. (45 min) as shown in FIG. 7, BLG wasreduced but its mobility in the gel was only slightly changed. Bycontrast, when incubated at 4° C. (17 h), the mobility of the bulk ofBLG was decreased significantly and a new form of the protein appeared(Lane 3, FIG. 7). An assessment of the extent of reduction by gelscanning revealed that BLG is partly reduced at 55° C. and fully reducedat 4° C. Comparison of the amino acid sequences of the tryptic peptidesof the partly and fully reduced forms with the known structure of BLGgave further information on the nature of the reduction.

BLG is known to contain two disulfides, both intramolecular (See, FIG.8): one clearly accessible on the surface and located close to theC-terminus (Cys 66-Cys 160) and the other, close to the core, locatedbetween two β-sheets (Cys 106-Cys 119 or possibly Cys 106-Cys 121).Sequence analysis confirmed that the exposed disulfide (Cys 66-Cys 160)was reduced at 55° C. (partly reduced form) and that this as well as thehidden disulfide (Cys 106-Cys 119 or Cys 106-Cys 121) were both reducedat 4° C. (fully reduced form) (see Materials and Methods). Differentialreduction of BLG could also be achieved by altering pH. Reduction at pH6.8 yielded only the partly reduced form whereas a mixture of the partlyand fully reduced forms was observed at pH 8.0 (both at 37° C.) (datanot shown). The results confirm the findings of others who showed thatBLG was unstable at pH values above neutrality (Tanford, C. et al.(1959), “Transformation of β-Lactoglobulin at pH 7.5”, Biochemistry81:4032–4036) and undergoes conformational transitions at temperaturesabove 40° C. (Qi, X. L. et al. (1997), “Effect of temperature on thesecondary structure of beta-lactoglobulin at pH 6.7, as determined by CDand IR spectroscopy: a test of the molten globule hypothesis”, Biochem.J. 324:341–346). Significantly, the same reduction results for BLG asshown in FIG. 7 were obtained when only the components of theNADP/thioredoxin system were added to raw milk without buffer.Furthermore, the BLG in this treated milk without buffer remainedreduced for 2 days when stored at 4° C. in air (data not shown).

As seen in Lanes 4 and 5 of the gel in FIG. 7, the monothiol glutathionemaintained in the reduced state by NADPH and glutathione reductase alsoreduced BLG and to some extent other milk proteins, but less effectivelythan NTS. Other disulfide reductants, dithiotreitol and lipoic acid,were effective but only when combined with thioredoxin as described inprevious Examples for venom neurotoxins (data not shown).

Digestion of Milk Proteins by Pepsin and Trypsin.

As shown in Examples 19 and 29, the trypsin sensitivity of smallproteins containing intramolecular disulfide bonds (e.g., trypsininhibitors, venom neurotoxins) increases dramatically followingreduction by thioredoxin. Likewise, BLG was seen to follow this samepattern, that is, the thioredoxin-reduced BLG was highly sensitive totrypsin digestion whereas the oxidized BLG (i.e., pure, untreated) wasresistant (See, Example 34). Similar results were obtained in thisExample with the pure BLG as well as with milk subjected to simulatedgastric fluid as described by Astwood, J. D. et al., supra. Whenseparated by SDS-PAGE and stained with Coomassie blue, BLG was found tobe digested by pepsin but only after reduction by thioredoxin (See, FIG.9). FIG. 9 shows the digestion of oxidized and reduced BLG as determinedby mini SDS/PAGE and Coomassie blue dye. All incubations with SGF wereat 37° C. 13.5 μl of SGF mixture was applied as indicated. The othervariables for the gel in FIG. 9 were: Lane 1, BLG, SGF, 0 min; Lane 2,BLG, SGF, 60 min; Lane 3, reduced BLG by NTS at 55° C., SGF, 0 min; Lane4, reduced BLG by NTS at 55° C., SGF, 60 min; Lane 5, reduced BLG at 4°C., SGF, 0 min; Lane 6, reduced BLG at 55° C., SGF, 60 min; Lane 7, SGF;Lane 8, BLG. As seen in FIG. 9, the difference in sensitivity wasstriking.

Oxidized BLG in milk was found to resist digestion for at least 60 minwhereas the thioredoxin-reduced form was digested within 60 seconds(See, FIGS. 10A–10C). FIGS. 10A, 10B and 10C depict the effect of timeon the digestion of milk buffered in PBS after thioredoxin reductiondetermined by mini SDS/PAGE and Coomassie blue dye. Samples 2–10contained 13.5 μl of simulated gastric fluid mixture and samples 3–10contained 50 μg milk protein. After 0, 0.25, 1, 2, 4, 8, 15, 30, 60 minincubation the digestion was stopped by neutralization and aliquots wereapplied to the appropriate lanes of each of the gels in FIGS. 10A, 10Band 10C. The other variables for the gel in FIG. 10A were buffered milkcontrol; for the gel in FIG. 10B: buffered milk, NTS, 55° C.; for thegel in FIG. 10C: buffered milk, NTS, 4° C. The reduction of a singledisulfide bond was sufficient. The partly (55° C.) and fully (4° C.)reduced forms of BLG showed no difference in pepsin sensitivity (compareFIGS. 10B and 10C). Significantly, even though partially reduced bythioredoxin, the glutathione-treated sample was insensitive to digestionby simulated gastric fluid (data not shown). While the sensitivity ofα-lactalbumin and κ-casein were somewhat enhanced by thioredoxinreduction, BLG, either pure or in milk, was found to be the only proteinnot digested without reduction by thioredoxin (See, FIGS. 7, 9 and10A–10C).

Allergy Status of Reduced and Digested Milk Proteins.

A comparison of the allergenicity of the major proteins of milk (casein,α-lactalbumin, BSA, BLG) using dog model skin tests revealed that BLG isthe major allergen of milk, accounting for 80% of the totalwheal-inducing activity. Furthermore, when treated with the thioredoxinsystem, both milk and BLG showed a decreased ability to elicit anallergic response. Thus, similar to the results in the previous Examplestesting allergenicity, the allergenicity of raw milk was decreased by afactor of 10 to 300, depending on the sensitivity of the dog tested(See, FIGS. 11A and 11B). The graphs in FIGS. 11A and 11B compare thethioredoxin-linked mitigation of skin test response to raw milkallergens in two dogs of differing sensitivity. The type Ihypersensitivity reaction determined by the wheal area (mm²) was inducedby 100 μl intradermal injections of milk solution in PBS as indicted inFIGS. 11A and 11B. The solutions were either pretreated with theNADP/thioredoxin system (NTS, 4° C.) or untreated (control). Theresponse of the two dogs is shown to illustrate that despite differencesin sensitivity, the thioredoxin treatment mitigated allergenicity inboth cases (i.e., for mildly (FIG. 11B) and highly (FIG. 11A) sensitiveanimals). A PBS/glycerol control and NTS were found to be negative foreach dog. Also, tests with a number of dogs showing differentsensitivity revealed no consistent difference in the allergenicity ofthe partly and fully reduced forms of BLG either pure or in milk (datanot shown). The skin test data thus show that reduction by thioredoxinalters epitope accessibility such that the allergenicity of BLG andpossibly other milk proteins is decreased.

Pure BLG showed an allergenic response similar to milk (compare oxidizedand reduced zero time samples in FIGS. 12A and 12B). In FIGS. 12A and12B, the oxidized “0” min and reduced “0” min bars represent the samplesbefore digestion treatment, the oxidized 60 min and reduced 60 min barsrepresent samples treated with pepsin containing SGF. The type Ihypersensitivity reaction observed by the skin wheal area (mm²) wasinduced by 100 μl intradermal injections of digested neutralized BLG(FIG. 12A) or milk (FIG. 12B) either pretreated with theNADP/thioredoxin system (NTS) (reduced) or untreated (oxidized control).A neutralized SGF control was found to be negative for all tested dogs.Furthermore, skin tests showed no difference between the pure A and Bforms of BLG with respect to the effect of thioredoxin on allergenicity(data not shown).

Skin tests carried out with both BLG and milk revealed that pepticdigestion nearly completely eliminated the allergenicity of bothpreparations (See FIGS. 12A and 12B). Based on skin tests, the allergyresponse is decreased by 300 to 1000 when digestion is coupled toreduction. Furthermore, the allergenicity of the digested samples wasdecreased to marginal levels, in both highly and mildly sensitive dogs.

These results are consistent with the conclusion that reduction bythioredoxin (1) alters the accessibility of the epitopes of intactproteins, so that allergenicity is decreased in most animals, and (2)renders stable allergens such as BLG susceptible to pepsin digestionwith the consequent almost complete loss of allergenic properties (seeFIGS. 13 and 14).

Finally, it was also found that α-lactalbumin and BLG are digested bytrypsin when reduced by the NTS system (See, Example 34). These results,coupled with the results obtained in this example with pepsin, confirmthe ability of thioredoxin to render BLG protease sensitive.

Feeding Trials with Atopic Dogs.

Upsets in the gastrointestinal tract leading to vomiting and diarrheaare symptoms that accompany the ingestion of food allergens but also theindigestibility of food proteins. Furthermore, the severity of thesesymptoms provides a measure of the strength of allergens thatcomplements skin tests. To obtain evidence on the gastrointestinalresponse, the food of sensitive dogs was supplemented with BLG. As shownfrom the results set forth in Table VIII below, dogs consuming foodcontaining 2.5 gm of reduced BLG (2.5 gm of BLG corresponds to ¾ literof milk) showed a significantly reduced extent of the vomiting. Repeatedfeeding trails indicated that, on average, about 70% of the gastricreflux disappeared. Significantly, this pattern was observed in a singleset of dogs fed either treated or untreated BLG. A similar alleviationof the associated gastrointestinal upset response was observed bydecreasing the BLG from the 2.5 gm used in Table VIII to 1.25 gm per canof food fed to each dog (data not shown). These observations complementthe skin test results discussed above in showing that allergenrecognition by the mucosal lymphatic tissue is mitigated by treatmentwith thioredoxin.

TABLE XVIII Allergic Response of Dogs Alternately Fed Untreated orThioredoxin-Treated β-Lactoglobulin Upper GI Index* Exp. A Exp. B DogControl Treated Control Treated 6GCB3 7 — — 0 6GCB7 6 — — 3 6GCB1 — 0 5— 6GCB4 — 6 9 — 6GCB6 — 0 8 — *Measure of quantity and fluidity ofinduced vomit Upper GI index response was measured using thioredoxintreated or untreated (control) BLG, 2.5 gm, mixed with one can of dogfood. Index calculations were performed after two separate feedingexperiments, A and B. Upper GI index quotations were: no vomit = 0,small vomit = 1, large vomit = 2, solid vomit = 1, liquid vomit = 2,liquid vomit with blood or bile = 3, delayed vomit = 1 and immediatevomit = 2.Discussion

In this Example, thioredoxin was found to reduce specifically theintramolecular disulfide bonds present in BLG, a major milk allergen.Depending on conditions, thioredoxin, reduced by NADPH and NTR reducedeither one or both of the disulfide bonds of BLG whether analyzed withpure protein or milk. The change in epitope distribution revealedexperimentally by skin tests and feeding challenges was seen at themolecular level in structural models (Peitsch, M. C. (1996), supra.).When the exposed disulfide bond of BLG (Cys66-Cys160) was disrupted bysite-directed mutagenesis using a computer model, the ¹²⁵Thr-¹³⁵Lysepitope (Kaminogawa, S. et al. (1989), “Monoclonal antibodies as probesfor monitoring the denaturation process of bovine β-lactoglobulin”,Biochemica et Biophisica Acta 998:50–56), which is nearby changed itsposition significantly whereas the epitope which is distant from bothdisulfides (⁸Lys—¹⁹Trp) did not (See, FIGS. 13 and 14). Mutagenesis ofthe buried disulfide (Cys106-Cys119 or possibly Cys106-Cys121) led to nofurther change in the position of the epitope. A similar observation wasmade with a model prepared with human BLG epitopes (Ball, G. et al.(1994), “A major continuous allergenic epitope of bovinebeta-lactoglobulin recognized by human IgE binding”, Clinical andExperimental Allergy 24:758–764).

The present results suggest that reduction by thioredoxin lowersallergenicity of a target protein allergen in two ways. Reductioneffects a change in protein structure that restricts epitopeaccessibility and enhances digestibility. In this way, the strength ofthe allergen is decreased and, in addition, should be more rapidlyeliminated in the gastrointestinal tract.

EXAMPLE 40 Digestion of Reduced Gliadins by Pepsin and Trypsin

The protease susceptibility of the gliadin fraction isolated by ethanolextraction of wheat flour as described in Examples 9 and 10 wasinvestigated. As shown in previous Example 10, gliadins containintramolecular disulfide bonds that are specifically reduced bythioredoxin. Gliadins are also a major food allergen in wheat. Buchanan,B. B. et al. (1997), “Thioredoxin-linked mitigation of allergicresponses to wheat”, Proc. Natl. Acad. Sci. USA 94:5372–5377. Thethioredoxin itself can be reduced either enzymatically by NADPH andNADP-thioredoxin reductase or chemically by dithiothreitol (DTT). Forthis Example, aliquots (50 μl) of isolated gliadin (1.04 mg/ml) wereincubated with or without thioredoxin (1.0 μg) which was reduced in thepresence or absence of 1 mM DTT for an hour at room temperature at pH7.5 in Tris-HCl buffer. At the end of the incubation, 5 μl of trypsinfrom Sigma (1 mg/ml) was added to each sample. The samples weresubjected to digestion for another hour at 30° C. Digestion wasterminated by PMSF (phenylmethylsulfonyl fluoride (2 μl of 100 mM) ineach sample. After mixing with SDS (10 μl of 10%) and glycerol (15 μl of50%), samples were analyzed by SDS-PAGE. After electrophoresis the 15%gel was stained with Coomassie blue and destained by methanol and aceticacid. Analysis of the protein bands demonstrated that the gliadins werestable to digestion in the oxidized (untreated) state but were degradedto lower molecular weight components following reduction by thioredoxin.Similar results were obtained when the gliadin aliquots were treated anddigested with pepsin instead of trypsin as in Example 39.

This Example and Examples 34 and 39 show that a consequence of allergenreduction by thioredoxin is the striking increase in sensitivity toproteases. Thus, whereas the oxidized forms of gliadins and BLG wereresistant to pepsin, the reduced forms were highly susceptible and werereadily digested. The average concentration of innate thioredoxin inwheat flour is about 0.01% (Johnson, T. C. et al. (1987), “Reduction ofpurothionin by the wheat seed thioredoxin system and potential functionas a secondary thiol messenger in redox control”, Plant Physiol.85:446–451). This is about 100 mg to about 200 mg per kilogram of flouror in bread, about 2 mg of thioredoxin per 100 gm of flour. However, theupper limit of naturally occurring thioredoxin in flour could be about 2mg per gram of food protein. Bread, for example, that contained higherconcentrations of thioredoxin treated with a thioredoxin reductant wouldbe a less allergenic and more digestible bread. The increase in thesensitivity to pepsin by food proteins treated with thioredoxin and athioredoxin reductant, seen also at the molecular level through modelingfor BLG, would likely lead to a more rapid processing of ingested BLG inthe gastrointestinal tract. If extended to the whole animal, treatmentof milk and wheat and other food products with the thioredoxin systemwould be expected to provide relief from the long-term effects ofallergenicity and indigestibility—notably edema and diarrhea.

Coummercialization of the thioredoxin technology with respect to milkcould be achieved by treating milk with the NTS before or afterpasteurization (i.e., at 55° C. for 45 min or at 4° C. for at least 10hr). Such applications might include the following:

-   -   1. Applying the thioredoxin system in liquid form to raw milk or        whey and coupling reduction to the pasteurization process.    -   2. Passing raw or pasteurized milk through a column of bound        reduced thioredoxin. The thioredoxin could be reduced with NADPH        and NTR prior to application of the milk. The thioredoxin could        also possibly be reduced with dithiothreitol which could be        removed from the column by washing prior to application of the        milk    -   3. Storing NTS treated milk under non-oxidizing conditions to        increase shelf life by using for example full or evacuated        containers.    -   4. Adding thioredoxin and NTR to milk after pasteurization and        adding NADPH in solid form just prior to use.    -   5. After treatment with thioredoxin, subjecting milk to limited        proteolytic digestion with enzymes such as trypsin. Such product        could be used to induce tolerance in milk-sensitive individuals.

EXAMPLE 41 Determination of the Decreased Allergenicity and IncreasedDigestibility of Thioredoxin Treated Ragweed Allergen Proteins

Reduction of pollen proteins by the thioredoxin system. Giant ragweedallergen extract, purchased from Bayer Inc., was analyzed for protein bythe Bradford assay using bovine γ-globulin as standard (Wong, J. H. etal. (1995) “Thioredoxin and seed proteins” Methods Enzymol 252:228–240).The protein, 100 μg, was reduced with 1.25 mM NADPH, 1.7 μg of E. coliNADP/thioredoxin reductase (NTR) and 1.7 μg of E. coli thioredoxin (Trx)by incubation for 45 min at 37° C. in 30 mM physiological bufferedsaline (PBS) (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ 2.7 mM KC1 and 137 mM NaCl,pH 7.4) resulting in a final volume of 100 μl. The extent of reductionwas determined by the SDS-PAGE/monobromobimane procedure as previouslydescribed above and also as described by Wong et al. (Ibid.). Gels werevisualized under U.V.

Skin tests. Procedures to measure the type I hypersensitivity reactionby skin tests in allergic doses were substantially the same to thosepreviously described above (see, Examples 36, 37 and 39). In brief,Evans blue dye 0.5% (0.2 ml/kg) was injected intravenously 5 minutesprior to skin testing. Aliquots of 0.1 ml of pollen allergen extractwere injected intradermally on ventral abdominal skin in half-logdilutions. Skin tests were read blindly by the same experienced blindedobserver scoring two perpendicular diameters for each blue spot.Appropriate negative controls (diluted in PBS) were included for eachanimal tested.

Digestion of pollen proteins by simulated gastric fluid. Pollen protein,650 μg, or pure Amb t V, 100 μg, was incubated in 100 μl PBS buffer at37° C. for 45 min, with or without a mixture of 2.4 μg Trx, 4.2 μg NTRand 2.5 mM NADPH. Then, 50 μl of each reaction mixture, containing 325μg pollen protein or 50 μg Amb t V, was digested in 100 μl simulatedgastric fluid (SGF) as described by Astwood et al. (Astwood, J. D. etal. (1996), “Stability of food allergens to digestion in vitro” NatureBiotechnology 14:1269–1273. SGF is composed of 0.32% porcine pepsin(w/v) and 30 mM NaCl adjusted to pH 1.2 with HCl. The digestion mixtureswere incubated at 37° C. and stopped at 0, 0.25, 1, 2, 4, 8, 15 or 60min with a 0.375 volume of 160 mM Na₂CO₃ to give a neutral pH. Proteinswere analyzed by SDS-PAGE and, as indicated, by skin tests.

Amb t V purification. Purification was achieved by adaptation of themethod of Roebber et al. 1985 (Roebber, M. et al. (1985),“Immunochemical and genetic studies of Amb.t. V (Ra5G), an Ra5 homologuefrom giant ragweed pollen” J. Immunol. 134:3062–9). In brief, 100 g ofnon-defatted pollen (Greer Laboratories) was suspended in 1 liter ofbuffer [50 mM Tris-HCl pH 7.4 containing 1 μM phenylmethylsulfonylfluoride (PMSF) and 1 mM EDTA-Na] and stirred gently for 30 min at roomtemperature. The mixture was then centrifuged for 15 min at 3,840×g, 4°C. The collected supernatant fraction was filtered once through glasswool and then twice through Whatman quantitative filters. The pelletcontaining the pollen grains was not further used. The high quantity oflipids in the filtered supernatant fraction was removed by extractionwith an equal volume of petroleum ether and centrifuged at 11,300×g for10 min, 4° C. The petroleum ether step was repeated 4 times. Theresulting clarified solution was concentrated by passage through anAmicon YM-3 membrane and separated on a Sephadex G-50F gel filtrationcolumn (2.1×90 cm) equilibrated and eluted with 20 mM Tris-HCl pH 7.5supplemented with 200 mM NaCl. The fractions containing 5 kDa proteinswere analyzed by 15% SDS-PAGE, combined and concentrated again with aYM-3 membrane. Ammonium sulfate was added to the concentrated proteinsto a final concentration of 2.6 M. The protein mixture then wasfractionated on a 1 ml HiTrap Phenyl Sepharose column (Pharmacia)equilibrated with 200 mM phosphate buffer pH 7.0 and eluted with a 50 mldecreasing gradient ranging from 2.5 to 0 M ammonium sulfate in thissame buffer. The pure Amb t V was recovered in a single peak atapproximately 0.8 M. Finally, the pure protein was dialyzed against 5 mMpotassium phosphate buffer, pH 7.0 and stored at −70° C. for furtherexperiments. Protein content was quantified using a molar extinctioncoefficient of 5800.

Data analysis. The statistical significance of the skin test resultsshowing the thioxedoxin-linked mitigation of pollen allergens wasdetermined by paired one-tailed, t-tests. The null hypothesis, whichassumes no difference in wheal area induced by untreated vs.thioredoxin-treated pollen proteins, was tested against the alternativehypothesis that the treatment resulted in mitigation of allergicresponse. The t-tests were completed for each dilution series (0.07 to219 ng allergen) at 0.05 level of significance on all sensitive dogs(df=8).

Results and Discussion

Reduction of the pollen proteins. As shown in FIG. 15, several proteinswere actively reduced by thioredoxin, namely Amb t V at 5 kDa, andunidentified proteins at 12, 14 and 35 kDa. Furthermore, the extent ofreduction varied with the reaction temperature. As shown in FIG. 15,reduction was most efficient at 37° C. or higher. In the case of Amb tV, the results indicate that multiple disulfide bonds (possibly all 4)are reduced at 37° C. and 55° C., whereas a lower number is reduced at4° C. In parallel, it was also observed that reduction by thioredoxinstrikingly affected the high thermostability described for Amb t V(Baer, H. et al. (1980), “The heat stability of short ragweed pollenextract and the importance of individual allergens in skin reactivity”J. Allergy Clin. Immunol. 66:281–5). Precipitation of protein was foundto occur at 55° C. after reduction by thioredoxin (data not shown).

Mitigation of the allergenicity by thioredoxin reduction. As shown inTable XIX below, reduction by thioredoxin effectively mitigated the skintest response. While less pronounced than found with milk (see, Example39), the thioredoxin-linked decrease in the allergenicity of pollencrude extract ranged between 3- and 33-fold. Furthermore, based on ttest analysis, the mitigation was statistically significant.

TABLE XIX Statistical evaluation of the mitigation by thioredoxin of theskin test response to pollen allergy. Paired one tail t test two samplesfor mean where P value is equal to or less than 0.05. Allergen,Observation, t test ng n t Critical value P value Mitigation 219 201.729 0.974 0.1711 Not Significant 66 27 1.706 2.326 0.0140 Significant22 27 1.706 3.045 0.026 Significant 6.6 27 1.706 3.253 0.0016Significant 2.2 27 1.706 2.185 0.0191 Significant 0.66 27 1.706 2.8050.0047 Significant 0.22 27 1.706 1.545 0.672 Not Significant 0.07 101.833 1.471 0.0877 Not Significant

Effect of thioredoxin reduction on the sensitivity of pollen proteins topepsin. To determine whether reduction by thioredoxin increases thedigestibility of pollen allergens, the oxidized and reduced forms ofpurified Amb t V protein were treated with SGF (Astwood, J. D. et al.(1996), “Stability of food allergens to digestion in vitro” NatureBiotechnology 14:1269–1273. 12). First, it was found that, likebeta-lactoglobulin, Amb t V was digested only when reduced by thethioredoxin system. The oxidized protein was resistant to pepsin for upto 60 min, consistent with its allergenic potential as shown in FIG. 16A(Astwood, J. D. (1996), supra). By contrast, the thioredoxin-reducedprotein almost completely disappeared after 2 minutes as shown in FIG.16B. This sensitivity to pepsin was substantiated by skin test dataobtained with crude pollen extracts tested with 8 dogs. It was observedthat the skin test reaction elicited by the oxidized pollen extract wasretained after digestion in 4 of these dogs (typified by the dogdesignated 6CGB1, see Example 39, “Food sensitization of atopic dogs”),indicating that the allergens were pepsin resistant as shown in FIG.17A. By contrast, when the preparation was reduced by thioredoxin, theallergic response declined markedly in these dogs. This confirmed thatallergens were disulfide proteins and consistent with the gel data thatthe proteins had then been digested. The 4 other dogs (typified by thedog designated 5CBB3 in FIG. 17B) showed a pronounced decrease inreaction when the oxidized (untreated) proteins were digested bySGF—i.e., the allergens most active were digested from the outset. Thisfinding indicated that the second group is less sensitive to disulfideproteins.

The data in FIG. 18 provide evidence that this differential response tountreated and thioredoxin-treated preparations resides in thesensitivity of a particular animal to disulfide protein allergens. Asseen for pure Amb t V in FIGS. 17A and 17B, the disulfide proteins incrude preparations (including Amb t V) were strongly resistant to pepsinunless reduced by the NADP-thioredoxin system (FIGS. 18A and 18B).Furthermore, the reduction temperature is important; digestion increasedprogressively as the reduction temperature increased from 4° C. to 37°C. to 55° C. Without reduction, the proteins in the 5 to 20 kDa rangewere not digested even after 60 incubation with SGF.

The above results are in accord with the conclusion that, in contrast tomilk in which the major allergen—β-lactoglobulin—is a disulfide protein(del Val, G. et al. (1999), “Thioredoxin Treatment IncreasesDigestibility and Lowers Allergerucity of Milk” J. of Allergy Clin.Immunol. In press), ragweed pollen contains a complex allergen mixtureconsisting of proteins both with and without disulfide bonds.

These results show that the NADP/thioredoxin system alleviates theallergic response to a pollen. It also shows that the importance ofactive disulfide proteins on complex mixtures of allergens can beassessed by comparing the pepsin sensitivity of oxidized andthioredoxin-reduced samples. The more important that disulfide proteinsare to the allergic response, the more effective thioredoxin is in thealleviation of that response.

EXAMPLE 42 Use of Thioredoxin Treated Ragweed Pollen Allergen forImmunotherapy

An animal that exhibits sneezing and coughing (bronchospasms) uponinhaling a specific amount of an aerosol of ragweed pollen protein Amb tV is the subject of this investigation. The Amb t V protein in theaerosol has not been treated with any reducing agents. The animal issubcutaneously injected with increasing doses of a clinical solutioncontaining 67 micrograms/cc of Amb t V which has been purified andincubated with thioredoxin, NADPH and NTR as described in Example 41.These subcutaneous injections are to determine the closest dose or endpoint at which a local allergic reaction to the treated Amb t V occurs.A wheal of about 5mm is observed to occur at the injection site with 10nanograms (ng) in 0. 15 cc. With 10 ng established as the end point, theanimal is then injected subcutaneously three days later with an amountof Amb t V one log below the end point or, in this case, 1 ng. Thisdosage corresponds to the first dose in the injection schedule set forthin the table below.

TABLE XX Dilution IV (blue) 1:10,000 First dose 0.15 cc 2nd dose 0.30 cc3rd dose 0.60 cc 4th dose 1.00 cc Dilution III (green) 1:1,000 5th dose0.15 cc 6th dose 0.25 cc 7th dose 0.35 cc 8th dose 0.50 cc 9th dose 0.75cc 10th dose 1.00 cc Dilution II (yellow) 1:100 11th dose 0.15 cc 12thdose 0.25 cc 13th dose 0.35 cc 14th dose 0.50 cc 15th dose 0.75 cc 16thdose 1.00 cc Dilution I (red) conc 1:10 17th dose 0.10 cc + 0.10 ccsaline 18th dose 0.15 cc + 0.15 cc saline 19th dose 0.20 cc + 0.20 ccsaline 20th dose 0.25 cc + 0.25 cc saline 21st dose 0.30 cc + 0.30 ccsaline

The first dose therefore is a 0.15 cc injection of a 1:10,000 dilutionof the Amb t V 67 micrograms/cc solution. The subject is observed for alocal reaction. Since no reaction is observed upon injection of thefirst dose, the animal is given the second injection of 0.30 cc of the1; 10,000 dilution or 2 ng. After three days, the third dose of 0.60 ccof the 1:10,000 dilution is subcutaneously administered. The injectionschedule is then followed with injections being given every three daysuntil the top dose tolerated by the subject is reached. The top dose isindicated by a large local skin reaction, i.e., a wheal larger than thesize of a silver dollar, and/or systemic symptoms such as urticaria(hives), sneezing, vomiting or a fall in blood pressure. The top dosewith this animal is the 21st dose but with another subject it could havebeen the 11th, the 15th, or the 18th, etc. Upon observing the top dose,the dosage is decreased one or two doses and this reduced dosage is heldas the new top dose. The animal is subsequently injected with the newtop dose every three to four weeks. Approximately six months afterreceiving the new top dose at three to four week intervals, the subjectanimal inhales the same amount of the aerosol of Amb t V that previouslycaused sneezing. No, or limited, sneezing or coughing is observed as aresult of this inhalation. The animal continues to receive the new topdose at the regular intervals and remains free or comparatively free ofsneezing and any other allergic reactions upon further inhalation ofnonreduced Amb t V.

EXAMPLE 43 Comparison Between Thioredoxin-Treated Pollen Protein andUntreated Pollen Protein for Immunotherapy Effectiveness

A group of 10 animals, all of the same species, that exhibited sneezingand other allergic reactions upon inhalation of a specific amount ofnon-reduced, allergen Amb t V are the subjects of the investigation. Todetermine an allergic end point, the animals are divided into two groupsof five each. The animals are tested for antibody levels and Group A isthen injected with increasing doses of non-reduced Amb t V, and Group Bis injected with increasing doses of thioredoxin, NADPH and NTRincubated Amb t V as described in Example 42. The doses based on mg/kgof body weight for all 10 animals is the same even though the absolutedosage given each animal may differ. The end point dose for each animalin the group is determined. The average end point dose based on mg/kg ofbody weight for Group A is lower than for Group B. The animals are theninjected according to the method and injection schedule set forth inExample 42. The top dose for each animal is determined. The top dosesfor the animals in Group B are consistently higher with lesser local orsystemic symptoms being observed than with the top doses for the animalsin Group A. Then in the manner described in Example 42, the animals areassigned a new top dose and are subsequently injected every three tofour weeks with this new top dose as in Example 42. Followingapproximately 6 months of such injections, the animals of Group A show asomewhat diminished allergic response upon subsequent inhalation of thespecific amount of non-reduced allergen, while animals of Group Bexhibit very limited or no significant allergic response. Furthermore,animals of Group B are able to tolerate much higher doses of theallergen, in some cases up to 10 to 100 times. A test to determine theantibody levels in the treated animals shows that the IgG antibody titerin the Group B animals is higher following immunotherapy while the titerof IgE is lower over time. The IgG antibody titer is also somewhathigher in Group A following immunotherapy, but much lower on the averagethan the IgG titer of the Group B animals.

These results show that

-   -   Thioredoxin reduction of ragweed pollen mitigates the allergic        response.    -   Thioredoxin reduction markedly enhances the digestibility of        disulfide pollen allergens.    -   Thioredoxin (and potentially other disulfide reductants such as        lipoic acid) is useful in a hypo-allergenic pollen extract for        immunotherapy of pollen allergic patients by enhancing the        production of IgG instead of IgE antibodies and improving the        safety of the therapy.

Embodiments of the invention could include treating pollen allergieswith eye drops or nose spray containing:

-   -   lipoic acid,    -   the NADP/thioredoxin system,    -   lipoic acid and the NADP/thioredoxin system.

This invention can be used to treat and prevent many types of allergies(e.g., food, pollen, dust mite) due to disulfide proteins by pretreatingthese proteins with the NADP/thioredoxin or lipoic acid as indicatedabove.

CONCLUDING REMARKS

It can be seen from the foregoing general description of the inventionand from the specific examples illustrating applications thereof, thatthe invention has manifold and far reaching consequences. The inventionbasically provides novel dough and dough mixtures and novel methods forcreating new doughs and for improving the quality of dough and bakedgoods as well as novel methods for inactivating enzyme inhibitors incereal products. The invention also provides a novel method for alteringthe biological activity and inactivity of animal toxins, and foreliminating or decreasing the allergenicity of several allergens, namelypollens and foods, namely wheat, egg, milk, whey, soy, nuts and beef.The invention further provides a method for increasing the proteolysisand digestibility of pollen and food allergens, particularly milk, wheyand their products and wheat products. In addition, the inventionprovides a novel protein that is a pullulanase inhibitor and a methodfor its inactivation.

While the invention has described in connection with certain specificembodiments thereof, it should be realized that various modifications asmay be apparent to those of skill in the art to which the inventionpertains also fall within the scope of the invention as defined by theappended claims.

1. A method of increasing the digestibility of a pollen proteincomprising: treating said pollen protein with an amount of thioredoxin,nicotinamide adenine dinucleotide phosphate-thioredoxin reductase (NTR)and reduced nicotinamide adenine dinucleotide phosphate (NADPH)effective for increasing the digestibility of said protein.
 2. Themethod of claim 1 wherein the amount of thioredoxin is about 0.1 mg to6.0 mg, the amount of NTR is about 0.01 mg to 8.0 mg and the amount ofNADPH is about 1.0 micromole to 500 micromoles per 1 gram of pollenprotein.
 3. The method of claim 1 wherein the amount of thioredoxin isabout 0.1 mg to 4.0 mg, the amount of NTR is about 0.1 mg to 7.0 mg andthe amount of NADPH is about 25 micromoles to 500 micromoles per gram ofprotein.
 4. The method of claim 1 wherein the amount of thioredoxin isat least about 0.01 mg, the amount of NTR is at least about 0.01 mg andthe amount of NADPH is at least about 1.0 micromole per grain ofprotein.
 5. The method of claim 1 wherein said pollen protein is a giantragweed pollen protein.
 6. The method of claim 5 wherein said pollenprotein is Amb t V.
 7. A method for decreasing the allergenicity of enanimal to an allergen protein having disulfide bonds comprising:reducing disulfide bonds in said specific protein by treating saidprotein with thioredoxin, nicotinamide adenine dinucleotidephosphate-thioredoxin reductase (NTR) and reduced nicotinamide adeninedinucleotide phosphate (NADPH).
 8. The method of claim 7 wherein theamount of thioredoxin is about 0.01 mg to 6.0 mg the amount of NTR isabout 0.01 mg to 8.0 mg and the amount of NADPH is about 1.0 micromoleto 500 micromoles per gram of said protein.
 9. The method of claim 7wherein the amount of thioredoxin is about 0.1 mg to 4.0 mg, the amountof NTR is about 0.1 mg to 7.0 mg and the amount of NADPH is about 25micromoles to 500 micromoles per gram of said protein.
 10. The method ofclaim 7 wherein the amount of thioredoxin is at least about 0.01 mg, theamount of NTR is at least about 0.01 mg and the amount of NADPH is atleast about 1.0 micromole per gram of said protein.
 11. The method ofclaim 7 wherein said allergen protein is a pollen protein.
 12. Themethod of claim 11 wherein said pollen protein is Amb t V.
 13. A methodof decreasing the allergenicity of a pollen protein containing disulfidebonds comprising incubating said pollen protein with an amount ofthioredoxin, NADPH and NTR effective for reducing the disulfide bonds ofsaid protein.